Alfalfa CRISPR/Cas9 genome editing system and application thereof
阅读说明：本技术 紫花苜蓿CRISPR/Cas9基因组编辑系统及其应用 (Alfalfa CRISPR/Cas9 genome editing system and application thereof ) 是由 王涛 董江丽 叶沁怡 于 2021-08-03 设计创作，主要内容包括：本发明公开了CRISPR/Cas9基因组编辑系统在紫花苜蓿基因编辑中的应用,所述CRISPR/Cas9基因组编辑系统包括表达载体,所述表达载体可包括Cas9表达盒、sgRNA1表达盒和sgRNA2表达盒,所述Cas9表达盒表达Cas9。所述sgRNA1表达盒表达名称为sgRNA1的sgRNA,所述sgRNA2表达盒表达名称为sgRNA2的sgRNA；所述sgRNA1表达盒含有名称为MtU6-6promoter的启动子和由所述MtU6-6promoter驱动的sgRNA1基因,所述sgRNA2表达盒含有名称为MtU6-5promoter的启动子和由所述MtU6-5promoter驱动的sgRNA2基因；所述sgRNA1和所述sgRNA2可针对紫花苜蓿的同一靶标基因或不同靶标基因。本发明根据紫花苜蓿同源四倍体的特点对靶标基因设计双靶点,构建基因编辑双元载体p6401-Target,由农杆菌介导转化紫花苜蓿获得再生植株。这种优化的基因组编辑系统大大提高了紫花苜蓿基因编辑效率,植株基因编辑效率可高达100％,单个靶点编辑效率可高达96.9％。(The invention discloses an application of a CRISPR/Cas9 genome editing system in alfalfa gene editing, wherein the CRISPR/Cas9 genome editing system comprises an expression vector, the expression vector can comprise a Cas9 expression cassette, a sgRNA1 expression cassette and a sgRNA2 expression cassette, and the Cas9 expression cassette expresses Cas 9. The sgRNA1 expression cassette expresses a sgRNA named sgRNA1, and the sgRNA2 expression cassette expresses a sgRNA named sgRNA 2; the sgRNA1 expression cassette contains a promoter named MtU6-6promoter and a sgRNA1 gene driven by the MtU6-6promoter, and the sgRNA2 expression cassette contains a promoter named MtU6-5promoter and a sgRNA2 gene driven by the MtU6-5 promoter; the sgRNA1 and the sgRNA2 can be directed against the same target gene or different target genes of alfalfa. The invention designs double targets for Target genes according to the characteristics of alfalfa autotetraploid, constructs a gene editing binary vector p6401-Target, and obtains regenerated plants by agrobacterium-mediated transformation of alfalfa. The optimized genome editing system greatly improves the alfalfa gene editing efficiency, the plant gene editing efficiency can reach 100%, and the single target point editing efficiency can reach 96.9%.)
The application of a CRISPR/Cas9 genome editing system in alfalfa genome editing is characterized in that the CRISPR/Cas9 genome editing system comprises an expression vector, the expression vector comprises a Cas9 expression cassette, an sgRNA1 expression cassette and an sgRNA2 expression cassette, the Cas9 expression cassette expresses a Cas9, an sgRNA1 expression cassette expresses an sgRNA with the name of sgRNA1, and the sgRNA2 expression cassette expresses an sgRNA with the name of sgRNA 2; the sgRNA1 expression cassette contains a promoter named MtU6-6promoter and a sgRNA1 gene driven by the MtU6-6promoter, and the sgRNA2 expression cassette contains a promoter named MtU6-5promoter and a sgRNA2 gene driven by the MtU6-5 promoter; the sgRNA1 and the sgRNA2 are directed against the same target gene or different target genes of alfalfa.
2. The use according to claim 1, wherein said MtU6-6promoter is a1) or a2) DNA molecule:
A1) the nucleotide sequence is a DNA molecule at the 1 st to 500 th site of the sequence 1 in the sequence table;
A2) a DNA molecule having 80% or more identity to the DNA molecule of A1) and having a promoter function;
the MtU6-5promoter is A3) or A4),
A3) the nucleotide sequence is a DNA molecule at the 789-1288 th site of the sequence 1 in the sequence table;
A4) a DNA molecule having 80% or more identity to the DNA molecule of A3) and having a promoter function.
3. The use of claim 1 or 2, wherein the sgRNA1 expression cassette and the sgRNA2 expression cassette each contain a terminator named AtU6-26t, wherein the AtU6-26t is B1) or B2):
B1) the nucleotide sequence is a DNA molecule at the No. 605-788 site of the sequence 1 in the sequence table;
B2) a DNA molecule having 80% or more identity to the DNA molecule of B1) and having a terminator function.
4. The use as claimed in any one of claims 1 to 3 wherein the nucleotide sequence of the sgRNA1 expression cassette is position 1 to 788 of sequence 1 in the sequence table, wherein the nucleotides 502 and 520 of sequence 1 are the same as the target sequence of the sgRNA 1; the nucleotide sequence of the sgRNA2 expression cassette is 789-1576 th of sequence 1 in the sequence table, and the 1290-1308 th nucleotides of the sequence 1 are identical to the target sequence of the sgRNA 2.
5. The use of any one of claims 1-4, wherein the sgRNA1 and the sgRNA2 are directed against the same target gene of alfalfa and are MsGA3ox1 gene, and the coding sequence (CDS) of the MsGA3ox1 gene is shown as sequence 2 in the sequence listing.
6. The use of claim 5, wherein the target sequence of the sgRNA1 is the reverse complement of nucleotides 347-365 of sequence 2 in the sequence table, and the target sequence of the sgRNA2 is nucleotides 341-359 of sequence 2 in the sequence table.
7. The use of any one of claims 1-4, wherein the sgRNA1 and the sgRNA2 are directed against the same target gene of alfalfa and are MsNP1 gene, and the coding sequence (CDS) of the MsNP1 gene is shown as sequence 3 in the sequence table.
8. The use of claim 7, wherein the target sequence of the sgRNA1 is the sequence 1052-1070 nucleotides of sequence 3 in the sequence table, and the target sequence of the sgRNA2 is the reverse complement of the sequence 1082-1100 nucleotides of sequence 3 in the sequence table.
9. The expression vector of any one of claims 1-8.
10. The use of the expression vector of claim 9 in the preparation of creeping alfalfa or in the preparation of male sterile alfalfa or the male sterile maintainer line alfalfa.
Technical Field
The invention relates to the technical field of biology, in particular to an alfalfa CRISPR/Cas9 genome editing system and application thereof.
Background
Alfalfa (Medicago sativa) is an important leguminous forage crop, has rich nutritional value, especially high protein content, contains high-quality dietary fiber and multiple vitamins, and is vegetatively reputed as the king of forage. Alfalfa is widely distributed in China, and has important functions on the protection and maintenance of grassland ecology besides excellent feeding value. Therefore, the physiological and ecological research and the genetic breeding work of the alfalfa have important practical significance. However, the alfalfa cultivars are mostly autotetraploids, and the genome is huge; the hybrid plant is a cross-bred plant and has the physiological characteristic of semi-self incompatibility; the heterozygosity of the genome is high, the genetic manipulation is difficult, and great difficulty is brought to basic research and breeding work.
The CRISPR/Cas9 genome editing technology is a powerful genetic manipulation tool, and mainly comprises a guide RNA (sgRNA) and a Cas9 protein. The sgRNA fused with the target sequence can be complementarily paired with the genome sequence, so that a gene editing machine is directionally guided to a target region, the DNA is cut by nuclease Cas9 to cause double-strand break, thereby causing DNA repair of an organism, and random mutation is introduced in the repair process, so that the target gene is edited. Although the alfalfa gene editing technology has been reported, the editing efficiency thereof cannot meet the requirements of practical application. Specifically, in the alfalfa gene editing system mediated by sgRNA driven by the alfalfa MtU6-1 promoter, the single target point editing efficiency reaches 52%, the homozygous mutation rate is 12%, and the editing efficiency has a great promotion space. In another literature report, the tRNA structure is used for serially connecting 4 gRNAs to carry out multi-target point editing on a single target gene of alfalfa, and the editing efficiency can reach 75% at most; the system has the defects that the arabidopsis U6 promoter sequence is still adopted to drive the expression of the tandem sgRNAs, the working efficiency of a single target point is still low, and the total editing of 4 copies of the alfalfa single gene can be realized only by connecting 4 sgRNAs in series. Therefore, improving the working efficiency of the CRISPR/Cas9 system in alfalfa is a key problem to be solved urgently by the alfalfa gene editing system.
Disclosure of Invention
The invention aims to solve the technical problem of how to quickly and efficiently realize the gene editing of tetraploid alfalfa.
In order to solve the technical problems, the invention provides an application of a CRISPR/Cas9 genome editing system in alfalfa gene editing, wherein the CRISPR/Cas9 genome editing system can comprise an expression vector, the expression vector can comprise a Cas9 expression cassette, a sgRNA1 expression cassette and a sgRNA2 expression cassette, the Cas9 expression cassette can express Cas9, the sgRNA1 expression cassette can express sgRNA named sgRNA1, and the sgRNA2 expression cassette can express sgRNA named sgRNA 2; the sgRNA1 expression cassette may contain a promoter named MtU6-6promoter and a sgRNA1 gene driven by the MtU6-6promoter, and the sgRNA2 expression cassette may contain a promoter named MtU6-5promoter and a sgRNA2 gene driven by the MtU6-5 promoter; the sgRNA1 and the sgRNA2 can be directed against the same target gene or different target genes of alfalfa.
The MtU6-6promoter and the MtU6-5promoter are both derived from medicago truncatula.
Further, in the application, the MtU6-6promoter can be the DNA molecule of A1) or A2):
A1) the nucleotide sequence is a DNA molecule at the 1 st to 500 th site of the sequence 1 in the sequence table;
A2) a DNA molecule having 80% or more identity to the DNA molecule of A1) and having a promoter function;
the MtU6-5promoter may be a DNA molecule of A3) or A4),
A3) the nucleotide sequence is a DNA molecule at the 789-1288 th site of the sequence 1 in the sequence table;
A4) a DNA molecule having 80% or more identity to the DNA molecule of A3) and having a promoter function.
Further, in the application, the sgRNA1 expression cassette and the sgRNA2 expression cassette both contain a terminator named AtU6-26t, and the AtU6-26t can be B1) or B2):
B1) the nucleotide sequence is a DNA molecule at the No. 605-788 site of the sequence 1 in the sequence table;
B2) a DNA molecule having 80% or more identity to the DNA molecule of B1) and having a terminator function.
In the above DNA molecules, the "identity" or "percentage of sequence identity" of the DNA is determined by comparing two optimally aligned sequences over a comparison window, where the optimal alignment provides the highest level of pairing and can introduce nucleotide additions among the test or reference sequences. Percent identity is determined by calculating the percentage of nucleotides at which the test and reference sequences are identical at each position throughout the sequence. Optimal sequence alignment and percent identity can be determined manually, or more preferably by computer algorithms including, but not limited to, TBLASTN, FASTA, GAP, BESTFIT, and CLUSTALW (Altschul et al, 1990, J.Mol.biol.215(3): 403-10; Pearson and Lipman,1988, Proc.Natl.Acad.Sci.USA 85(8): 2444-8; Thompson et al, 1994, Nucleic Acids Res.22(22): 4673-80; Deverux et al, 1984, Nucleic Acids Res.12: 387-395; Higgins et al, 1996, Methods Enzymol.266: 383-402). Preferably, NCBI Blast Server (http:// www.ncbi.nlm.nih.gov), set at default parameters, is used to search multiple databases for homologous sequences.
In the above DNA molecule, the 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
Further, in the application, the nucleotide sequence of the sgRNA1 expression cassette is 1 st to 788 th of a sequence 1 in a sequence table, wherein the nucleotides 502-520 th of the sequence 1 in the sequence table are identical to a target sequence of the sgRNA 1; the nucleotide sequence of the sgRNA2 expression cassette is 789-1576 th of a sequence 1 in a sequence table, and the 1290-1308 th nucleotide of the sequence 1 in the sequence table is identical to a target sequence of the sgRNA 2.
Specifically, the nucleotide sequence of the sgRNA1 expression cassette is shown in the 1 st-788 th position of the sequence 1 in the sequence table, the 1 st-500 th position of the sequence 1 in the sequence table is MtU6-6promoter, the 501 st position is the transcription initiation site nucleotide G, the 502 nd-520 th position nucleotide is the same as the target sequence of the sgRNA1, the 521 nd-604 th position nucleotide is sgRNA1 scaffold, and the 605 th-788 th position nucleotide sequence is AtU6-26 terminator.
Specifically, the nucleotide sequence of the sgRNA2 expression cassette is represented by 789-1576 of sequence 1 in the sequence table, the 789-1288-position of the sequence 1 in the sequence table is MtU6-5promoter, the 1289-position is the transcription initiation site nucleotide G, the 1290-position 1308-position nucleotide is identical to the target sequence of the sgRNA2, the 1309-1392-position nucleotide is sgRNA2scaffold, and the 1393-position 1576-position nucleotide sequence is AtU6-26 terminator.
Further, in the application, the sgRNA1 and the sgRNA2 can be the MsGA3ox1 gene of the same target gene of alfalfa, and the coding sequence of the MsGA3ox1 gene is shown as a sequence 2 in a sequence table.
Further, for the application, the target sequence of the sgRNA1 can be the reverse complement of nucleotide 347-365 of the sequence 2 in the sequence table (i.e. 5'-ACACCATCCGGGGAACGAA-3'), and the target sequence of the sgRNA2 can be nucleotide 341-359 of the sequence 2 in the sequence table (i.e. 5'-AAGCCATTCGTTCCCCGGA-3').
Further, in the application, the sgRNA1 and the sgRNA2 can be the MsNP1 gene of the same target gene of alfalfa, and the coding sequence of the MsNP1 gene is shown as a sequence 3 in a sequence table.
Further, for the application, the target sequence of the sgRNA1 can be the nucleotides 1052 and 1070 (i.e. 5'-GCTACATTGAAGCCGCTAG-3') of the sequence 3 in the sequence table, and the target sequence of the sgRNA2 can be the reverse complement of the nucleotides 1082 and 1100 (i.e. 5'-TCTCTTTGAGAACCTGTGA-3') of the sequence 3 in the sequence table.
The invention provides the expression vector applied to the application.
The invention provides application of the expression vector in preparing creeping alfalfa or male sterile alfalfa or alfalfa of the male sterile maintainer line.
The invention aims to provide an optimized alfalfa CRISPR/Cas9 genome editing method so as to efficiently obtain alfalfa mutant materials with all mutated alleles. 2U 6 promoters cloned from medicago truncatula are adopted to drive the expression of sgRNA, double targets are designed for Target genes according to the characteristics of alfalfa autotetraploids, a gene editing binary vector p6401-Target is constructed, and the medicago sativa is subjected to agrobacterium-mediated transformation to obtain a regenerated plant. The optimized genome editing system greatly improves the gene editing efficiency of the alfalfa, the gene editing efficiency of the plants reaches 100%, and the single target point editing efficiency can reach 96.9%.
Drawings
Fig. 1 is a vector structure diagram of an sgRNA module and a schematic diagram of a construction method of a sgRNA module Target-5CBC with double targets.
FIG. 2 is a schematic diagram of the structure of a binary vector p6401-Target for gene editing.
FIG. 3 shows the structure of the vector of alfalfa p6401-MsGA3ox 1.
FIG. 4 shows the result of identifying the transgene of alfalfa transferred into p6401-MsGA3ox1 vector to obtain regenerated plant.
FIG. 5 shows the mutation pattern analysis of the gene editing plant Msga3ox 1.
FIG. 6 shows the phenotype of Msga3ox1 mutant and wild type plants and associated statistical analysis.
FIG. 7 shows the structure of the vector of alfalfa p6401-MsNP 1.
FIG. 8 shows the result of identifying the transgene of alfalfa transferred into p6401-MsNP1 vector to obtain regenerated plants.
FIG. 9 shows the cloning result of target fragments of regenerated plants obtained by transferring alfalfa into p6401-MsNP1 vector.
FIG. 10 shows the sequencing result of the alfalfa transferred into p6401-MsNP1 vector positive plant target segment Sanger.
Fig. 11 is an example of the mutation pattern of the Msnp1 mutant.
Fig. 12 is a close-up of the inflorescence of the Msnp1 mutant and Control material (Control).
FIG. 13 shows a close-up of the male and female stamens of the Mspp 1 mutant and the Control material (Control).
FIG. 14 shows the results of the Alexander staining experiments with the Mspp 1 mutant and the Control material (Control).
FIG. 15 shows the results of the Mspp 1 mutant and Control material (Control) I2-KI staining experiments.
FIG. 16 shows the application of male sterile line of alfalfa.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
p6401 vector and p5 CBC: described in "ZHU, F., YE, Q., CHEN, H., DONG, J. & WANG, T.2021.multigene recording results at MtCEP1/2/12 reduced regulated latex root and non number in medical guide canal.J.Exp.Box," publicly available from the applicant and only available for duplicate invention experiments.
Bulk mother liquor (1L) 10 xn 6: MgSO (MgSO)4·7H2O 1.85g，KNO3 28.3g，(NH4)2SO4 4.63g，CaCl2·2H2O 1.66g，KH2PO4 4g。
1000 × SH minimal mother liquor (100 mL): MnSO4·H2O 1g，H3BO3 500mg，ZnSO4·7H2O 100mg，KI 100mg，Na2MoO4·2H2O 10mg，CuSO4·5H2O 20mg，CoCl2·6H2O 10mg。
1000 × SH organic mother liquor (100 mL): 500mg of nicotinic acid, 500mg of pyridoxine hydrochloride and 500mg of thiamine hydrochloride.
50 XEDFS iron salt mother liquor (500 mL): 3.487g of NaFe EDTA.
The formula of SH9 culture medium (1L) is as follows: 100mL of 10 XN 6 bulk mother liquor, 1mL of 1000 XSH micro mother liquor, 1mL of 1000 XSH organic mother liquor, 20mL of 50 XEDFS ferric salt mother liquor, 100mg of inositol and 20g of sucrose, and the pH is adjusted to 5.85. To the solid medium was added 8g of agar.
The formulation of SM4 medium (1L) was: MURASHIGE&SKOOG(MS)BASAL MEDIUM(M519)(Phyto Technology LaboratoriesTM)4.43g, 0.4mL of 2, 4-D stock solution (10mg/mL), 0.2mL of 6-BAP stock solution (1mg/mL), 30g of sucrose, and pH adjusted to 5.85. 3.2g of plant gel was added to the solid medium.
The formulation of MSBK medium (1L) was: MURASHIGE&SKOOG(MS)BASAL MEDIUM(M519)(Phyto Technology LaboratoriesTM)4.43g, 1mL of kinetin (1mg/mL), 0.5mL of 6-BAP stock solution (1mg/mL), 30g of sucrose, and pH adjusted to 5.85. 3.2g of plant gel was added to the solid medium.
1/2 the formulation of MS medium (1L) was: MURASHIGE&SKOOG(MS)BASAL MEDIUM(M519)(Phyto Technology LaboratoriesTM)2.22g, sucrose 12g, pH adjusted to 5.85. To the solid medium was added 8g of agar.
The data were processed in the following examples using SPSS16.0 statistical software and the results were expressed as mean ± standard deviation, with Kruskal-Wallis, nonparametric test, P < 0.05 (x) indicating a significant difference, P < 0.01 (x) indicating a very significant difference and P < 0.001 (x) indicating a very significant difference.
Summary of The Invention
The invention utilizes a CRISPR/Cas9 genome editing system to edit the alfalfa genome, and mainly comprises the following steps:
constructing an alfalfa gene editing vector p 6401-Target;
step (2) agrobacterium tumefaciens-mediated genetic transformation of alfalfa leaf discs;
and (3) identifying the genotype and phenotype of the alfalfa regenerated seedlings.
The construction of the alfalfa gene editing vector p6401-Target in the step (1) comprises the following aspects:
1. designing a gene editing target: according to the genome sequence of the alfalfa target gene, two specific target spots 1 and 2 are designed to ensure that the target gene can be efficiently edited.
2. Construction of sgRNA module: synthesizing primers with target1 and target2 respectively comprises
Target 1-BsF: 5 '-ATATATGGTCTCGCTTGNNNNNNNNNNNNNNNNNNNGTT-3', wherein the 18 th to 36 th nucleotides are designed target1 sequence;
target 1-F0: 5 '-GNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC-3', wherein the 2 nd to 20 th nucleotides are designed target1 sequence;
target 2-R0: 5 '-AACNNNNNNNNNNNNNNNNNNNCAATTTAATGGTTCGCTTGTA-3', wherein nucleotides 4 to 22 are the reverse complement of the designed target 2;
target 2-BsR: 5 '-ATTATTGGTCTCGAAACNNNNNNNNNNNNNNNNNNNC-3', wherein position 18 to position 36 are the reverse complement of the designed target 2;
the sgRNA module Target-5CBC with the Target sequence is obtained by bridging PCR amplification (as shown in figure 1), and the reaction system is as follows: 10 XKOD plus Buffer 5. mu. L, MgSO4(25mM), 3. mu. L, dNTPs (2mM, Toyobo) 4. mu.L, KOD plus (Toyobo) 1. mu. L, p5CBC (5 ng/. mu.L), 1. mu. L, Target1-BsF (20. mu.M), 1. mu. L, Target1-F0 (1. mu.M), 1. mu. L, Target2-R0 (1. mu.M), 1. mu. L, Target2-Bsr (20. mu.M), 1. mu. L, ddH2O 32. mu.L, and a total reaction volume of 50. mu.L.
The reaction procedure is as follows: a first round: denaturation at 94 deg.C for 2 min; and a second round: denaturation at 94 ℃ for 15sec, annealing at 60 ℃ for 30sec, extension at 68 ℃ for 1min, 30 cycles; and a third round: extension at 68 ℃ for 5 min. After the reaction is finished, detecting the product by 1% agarose gel electrophoresis, cutting a gel block where the target fragment is located, and recovering the fragment by using a gel recovery kit, wherein the size of the product fragment is 841 bp.
3. Construction of a binary vector for gene editing: the Target-5CBC fragment with the Target sequence and the p6401 vector are cut by restriction endonuclease BsaI by utilizing Golden Gate reaction, and are connected by T4 DNA ligase at the same time to form p6401-Tatget (shown in figure 2). The specific reaction system is as follows: target-5CBC fragment (. about.100 ng/. mu.L) 8. mu. L, p6401 (. about.400 ng/. mu.L) 2. mu.L, 10 XT 4 Ligase Buffer (NEB) 1.5. mu.L, 10 XBA 1.5. mu. L, BsaI (NEB)1. mu. L, T4 DNA Ligase (NEB)1. mu.L, total reaction volume 15. mu.L. The reaction procedure is as follows: the first step is as follows: 5h at 37 ℃; the second step is that: 50 ℃ for 5 min; the third step: 80 ℃ for 10 min.
After the reaction is finished, the product is added into escherichia coli TOP10 competent cells, ice bath is carried out for 30min, heat shock is carried out for 90s at 42 ℃, LB liquid culture medium without antibiotic is added, shaking culture is carried out at 37 ℃ and 150rpm for 45min, then bacterial liquid is coated on LB solid culture medium containing Kanamycin (50mg/L), and dark inversion screening culture is carried out at 37 ℃ for 12 h. Single clones are picked to 400 mu L LB liquid culture medium containing Kanamycin (50mg/L) and are shaken at 37 ℃ and 230rpm for 6-8 h, then bacterial liquid PCR identification is carried out, amplification is carried out by taking the Target1-BsF and the Target2-BSR as primers, and positive clones have specific bands with the size of 841 bp. Extracting positive cloning plasmid, and determining the sequence of the constructed transformation vector p6401-Target by sequencing, wherein the sequencing primers are Target1-BsF and Target 2-Bsr.
The sequencing result shows that: the alfalfa gene editing vector p6401-Target contains an sgRNA1 expression cassette and an sgRNA2 expression cassette, and the nucleotide sequence is a sequence 1, wherein n is a, t, c or g.
The nucleotide sequence of the sgRNA1 expression cassette is 1 st to 788 th of sequence 1 in the sequence table, wherein, the 1 st to 500 th of the sequence 1 in the sequence table is MtU6 to 6promoter, the 501 th is transcription initiation site nucleotide G, the 502 th and 520 th positions are the same as the target sequence of the sgRNA1, the 521 th and 604 th nucleotides are sgRNA1 scaffold, and the 605 th and 788 th nucleotide sequences are AtU6 to 26 terminator.
The nucleotide sequence of the sgRNA2 expression cassette is the 789-1576 th position of the sequence 1 in the sequence table, wherein the 789-1288 th position of the sequence 1 in the sequence table is MtU6-5promoter, the 1289 th position is the transcription initiation site nucleotide G, the 1290-1308 th position is the same as the target sequence of the sgRNA2, the 1309-1392 th position nucleotide is sgRNA2scaffold, and the 1393-1288 th position nucleotide sequence is AtU6-26 terminator.
The genetic transformation of the alfalfa leaf disc mediated by the agrobacterium tumefaciens in the step (2) comprises the following aspects:
1. transferring the gene editing binary vector p6401-Target into agrobacterium tumefaciens EHA 105: adding 50ng of plasmid into the competent cell of Agrobacterium tumefaciens EHA105, and carrying out ice bath for 5 min; adding the mixture into a precooled electric shock cup, carrying out high-voltage electric shock at 1600V, then adding 500 mu L of YEP liquid culture medium without antibiotics, blowing and uniformly mixing, transferring the bacterial liquid into a centrifuge tube, shaking at 28 ℃ and 200rpm for 1h, taking 30 mu L of bacterial liquid, coating the bacterial liquid on YEP solid screening culture medium containing Rifamicin (75mg/L) and Kanamycin (50mg/L), carrying out dark inversion screening and culture at 28 ℃ for 24-48 h, picking a single clone for colony PCR identification, carrying out amplification reaction by taking Target1-BsF and Target2-BSR in the step (1) as primers, selecting a positive clone with a specific band with the size of 841bp, and carrying out next infection by the selected positive clone.
2. Preparing an explant: taking the 5 th to 8 th fully-unfolded compound leaves of the alfalfa branch as an explant, which are taken as the receptor materials and counted from the top bud downwards, immersing the collected compound leaves in 0.1% Triton X100 aqueous solution for 5min, and washing the compound leaves for 3-5 times by using deionized water; and then transferring to a sterilized sealed bottle, adding 30% bleaching water (the effective component is 0.5% NaClO) for sterilization for 10min, and cleaning for later use in an ultra-clean workbench for 3-5 times by using the sterilized water.
3. Preparing an agrobacterium infection solution: the Agrobacterium tumefaciens EHA105 positive colonies transferred to p6401-Target were transferred to 200mL of YEP liquid medium containing Rifamicin (75mg/L) and Kanamycin (50mg/L) and shaken at 28 ℃ and 230rpm to OD600nm0.2 to 0.4. Transferring the bacterial liquid into a sterile centrifuge bottle, centrifuging at room temperature and 5000rpm for 10min, removing supernatant, and resuspending with 200mL of SM4 liquid culture medium (containing 0.1mM acetosyringone) to obtain the final product.
4. Agrobacterium-mediated genetic transformation and co-cultivation: putting the explant into the prepared staining solution, vacuumizing to-0.09 MPa for 5min, and slowly deflating; performing ultrasonic treatment for 3min at 20 ℃ in an ultrasonic cleaner; vacuumizing again to-0.09 MPa for 5min, and slowly deflating; removing the staining solution in a superclean workbench, and sucking the redundant liquid on the surface of the explant by using filter paper; the compound leaves were cut with a knife, the petioles were removed, and the cut leaves were plated on SM4 solid medium (containing acetosyringone 0.1mM) and incubated at 22 ℃ in the dark for 3 days.
5. Callus induction and differentiation: transferring the explants to SM4 solid medium (containing 200mg/L of timentin) containing the screening antibiotic hygromycin B (10mg/L) to induce resistant callus, culturing at 22 ℃, 16h of light and 8h of dark, subculturing once every 2 weeks for 6 weeks; then transferring the callus to MSBK solid medium (containing 200mg/L of timentin) containing screening antibiotic hygromycin B (10mg/L) to induce bud differentiation, culturing at 22 ℃, 16h of light and 8h of dark for 3 weeks; transferring the embryogenic callus to SH9 solid medium (containing 200mg/L of timentin) containing screening antibiotic hygromycin B (5mg/L) to continuously induce bud differentiation, culturing at 22 ℃ for 16h under light and 8h in the dark, and subculturing once every 3 weeks until regeneration seedlings are generated; transferring the regenerated seedlings to 1/2MS solid culture medium without antibiotics for rooting, subculturing once every 3-4 weeks, and culturing at 22 ℃, 16h of light and 8h of dark; after rooting, transferring the seedlings into nutrient soil, covering the seedlings with a preservative film for moisture preservation and culture for 5 days, and then uncovering the film, wherein the greenhouse condition is 22 ℃, 16 hours of illumination, 8 hours of darkness and 70-80% of humidity for culture.
The genotype identification of the alfalfa regeneration seedlings in the step (3) comprises the following aspects:
1. and (3) transgene identification: and taking a single leaf of the regenerated seedling to extract genome DNA, and then carrying out PCR detection. Plasmid p6401-Target is used as a positive control, receptor material genome DNA is used as a negative control, primers are Target1-BsF and Target2-BSR for amplification, and the reaction system is as follows: 2 XTaq mix 10 mu L, Target1-BsF 1 mu L, Target2-Bsr 1 mu L, template DNA 1 mu L, ddH2O7. mu.L, total volume of reaction was 20. mu.L.
The reaction procedure is as follows: a first round: denaturation at 95 deg.C for 3 min; and a second round: denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1min, 35 cycles; and a third round: extension at 72 ℃ for 5 min. After the reaction is finished, the product is detected by 1% agarose gel electrophoresis, the size of the product fragment is 841bp, and if a specific band with a corresponding size exists, the sample is positive for transgenosis.
2. And (3) mutation mode identification: designing specific primers in genome sequences near target gene targets 1 and 2 for amplification and Sanger sequencing, and performing preliminary mutation prediction according to a sequencing peak diagram by taking a receptor material as a control; the sample target fragments that were likely to be edited were ligated into the pMD18T vector, and multiple single clones were picked for sequencing validation.
Example 1 editing alfalfa MsGA3ox1 Gene with optimized CRISPR/Cas9 System to obtain dwarf plant type Material
Construction of Gene editing vector p6401-MsgA3ox1
1. Gene editing target design
The alfalfa MsGA3ox1 contains a coding sequence (CDS) which is the MsGA3ox1 coding gene shown in a sequence 2 in a sequence table. (ii) predicting websites by online target points based on the genomic sequence of alfalfa MsGA3ox 1: (http:// crispor.tefor.net/) And generating a target point list, and selecting target point 1 as 5 ' -ACACCATCCGGGGAACGAA-3 and target point 2 as 5'-AAGCCATTCGTTCCCCGGA-3'.
Construction of sgRNA Module
Synthesis of primers from Beijing Liuhe Hua Dageney science and technology Co
MsGA3ox1-Target1-BsF：5′-ATATATGGTCTCGCTTGACACCATCCGGGGAACGAAGTT-3', wherein the 18 th to 36 th nucleotides are designed MsGA3ox1 target1 sequence;
MsGA3ox1-Target1-F0：
5′-GACACCATCCGGGGAACGAAGTTTTAGAGCTAGAAATAGC-3', wherein nucleotides 2 to 20 are a designed MsGA3ox1 target1 sequence;
MsGA3ox1-Target2-R0：
5′-AACTCCGGGGAACGAATGGCTTCAATTTAATGGTTCGCTTGTA-3', wherein nucleotides 4 to 22 are the reverse complement of the designed MsGA3ox1 target 2;
MsGA3ox1-Target2-BsR：5′-ATTATTGGTCTCGAAACTCCGGGGAACGAATGGCTTc-3', wherein position 18 to position 36 are the reverse complement of the designed MsGA3ox1 target 2;
and performing bypass PCR amplification to obtain a sgRNA module MsGA3ox1-5CBC with a target sequence.
The sequence of the gel recovery fragment for sequencing MsGA3ox1-p5CBC is shown as a sequence 4 in the sequence table. The 18 th to 36 th positions of the sequence 4 are the target1 sequence of MsGA3ox1, and the 806 th and 824 th positions are the target2 sequence of MsGA3ox 1.
3. Construction of a binary vector for gene editing: the MsGA3ox1-5CBC fragment with the target sequence and the p6401 vector are cut by restriction enzyme BsaI and are connected by T4 DNA ligase by utilizing Golden Gate reaction to form p6401-MsGA3ox1 (shown in figure 3). After the reaction is finished, the product is added into escherichia coli TOP10 competent cells, ice bath is carried out for 30min, heat shock is carried out for 90s at 42 ℃, LB liquid culture medium without antibiotic is added, shaking culture is carried out at 37 ℃ and 150rpm for 45min, then bacterial liquid is coated on LB solid culture medium containing Kanamycin (50mg/L), and dark inversion screening culture is carried out at 37 ℃ for 12 h. Single clones are picked to 400 mu L LB liquid culture medium containing Kanamycin (50mg/L) and are shaken at 37 ℃ and 230rpm for 6-8 h, then bacterial liquid PCR identification is carried out, amplification is carried out by using the MsGA3ox1-Target1-BsF and MsGA3ox1-Target2-BSR as primers, and positive clones have specific bands with the size of 841 bp. Extracting positive clone plasmids, and determining the sequence of the constructed transformation vector p6401-MsGA3ox1 through sequencing, wherein sequencing primers are the primers MsGA3ox1-Target1-BsF and MsGA3ox1-Target 2-BsR.
Sequencing results show that p6401-MsGA3ox1 is a recombinant expression vector obtained by replacing the fragment between BsaI restriction enzyme cleavage sites of vector p6401 with the base sequence shown at positions 14-824 of fragment sequence 4, and keeping the other sequences of vector p6401 unchanged. p6401-MsGA3ox1 expressed two sgrnas targeting sgRNA1 (target 1) and sgRNA2 (target 2) of the MsGA3ox1 gene.
Second, agrobacterium tumefaciens mediated genetic transformation of alfalfa leaf discs
1. The gene editing binary vector p6401-MsGA3ox1 was transferred into Agrobacterium tumefaciens EHA 105: adding 50ng of plasmid into the competent cell of Agrobacterium tumefaciens EHA105, and carrying out ice bath for 5 min; adding the mixture into a precooled electric shock cup, carrying out high-voltage electric shock at 1600V, then adding 500 mu L of YEP liquid culture medium without antibiotics, blowing and uniformly mixing, transferring the bacterial liquid into a centrifuge tube, shaking and culturing at 28 ℃ and 200rpm for 1h, taking 30 mu L of bacterial liquid, coating the bacterial liquid on YEP solid screening culture medium containing Rifampicin (75mg/L) and Kanamycin (50mg/L), carrying out dark inversion screening and culturing at 28 ℃ for 24-48 h, selecting a single clone for colony PCR identification, carrying out amplification reaction by taking MsGA3ox1-Target1-BsF and MsGA3ox1-Target2-BSR in the step (1) as primers, selecting a positive clone (named as EHA105/p6401-MsGA3ox1) with a specific band with the size of 841bp, and selecting the positive clone for further infection.
2. Preparing an explant: taking the 5 th to 8 th fully-unfolded compound leaves of the alfalfa branch as an explant, which are taken as the receptor materials and counted from the top bud downwards, immersing the collected compound leaves in 0.1% Triton X100 aqueous solution for 5min, and washing the compound leaves for 3-5 times by using deionized water; and then transferring to a sterilized sealed bottle, adding 30% bleaching water (the effective component is 0.5% NaClO) for sterilization for 10min, and cleaning for later use in an ultra-clean workbench for 3-5 times by using the sterilized water.
3. Preparing an agrobacterium infection solution: EHA105/p6401-MsGA3ox1 was transferred to 200mL YEP broth containing Rifamicin (75mg/L) and Kanamycin (50mg/L) and shaken at 230rpm at 28 ℃ until the OD600 was 0.2-0.4. Transferring the bacterial liquid into a sterile centrifuge bottle, centrifuging at room temperature and 5000rpm for 10min, removing supernatant, and resuspending with 200mL of SM4 liquid culture medium (containing 0.1mM acetosyringone) to obtain the final product.
4. Agrobacterium-mediated genetic transformation and co-cultivation: putting the explant into the prepared staining solution, vacuumizing to-0.09 MPa for 5min, and slowly deflating; performing ultrasonic treatment for 3min at 20 ℃ in an ultrasonic cleaner; vacuumizing again to-0.09 MPa for 5min, and slowly deflating; removing the staining solution in a superclean workbench, and sucking the redundant liquid on the surface of the explant by using filter paper; the compound leaves were cut with a knife, the petioles were removed, and the cut leaves were plated on SM4 solid medium (containing acetosyringone 0.1mM) and incubated at 22 ℃ in the dark for 3 days.
5. Callus induction and differentiation: transferring the explants to SM4 solid medium (containing 200mg/L of timentin) containing the screening antibiotic hygromycin B (10mg/L) to induce resistant callus, culturing at 22 ℃, 16h of light and 8h of dark, subculturing once every 2 weeks for 6 weeks; then transferring the callus to MSBK solid medium (containing 200mg/L of timentin) containing screening antibiotic hygromycin B (10mg/L) to induce bud differentiation, culturing at 22 ℃, 16h of light and 8h of dark for 3 weeks; transferring the embryogenic callus to SH9 solid culture medium (containing 200mg/L of timentin) containing screening antibiotic hygromycin B (5mg/L) to continuously induce bud differentiation, culturing at 22 ℃ for 16h in the light and 8h in the dark, and subculturing once every 3 weeks until regeneration seedlings are generated; transferring the regenerated seedlings to 1/2MS solid culture medium without antibiotics for rooting, subculturing once every 3-4 weeks, and culturing at 22 ℃, 16h of light and 8h of dark; after rooting, transferring the seedlings into nutrient soil, covering the seedlings with a preservative film for moisture preservation and culture for 5 days, and then uncovering the film, wherein the greenhouse condition is 22 ℃, 16 hours of illumination, 8 hours of darkness and 70-80% of humidity for culture.
Third, genotype identification of alfalfa regenerated seedlings
In the rooting culture process of the genetic transformation of the alfalfa, the existence of regenerated seedlings with obviously dwarf plants is found, the phenotype is similar to that of the Mtga3ox1 mutant in the diploid medicago truncatula, and the gene editing possibly and successfully occurs is presumed according to the phenotype indicator. Therefore, partial dwarf seedlings and normal seedlings are selected for genotype identification.
1. And (3) transgene identification: and (3) extracting genome DNA from a single leaf of the regenerated seedling, and carrying out PCR detection by using the extracted genome DNA as template DNA. Plasmid p6401-MsGA3ox1 is used as a positive control, wild type genome DNA is used as a negative control, primers are MsGA3ox1-Target1-BsF and MsGA3ox1-Target2-BSR for amplification, and the reaction system is as follows: 2 XTaq mix 10 mu L, MsGA3ox1-Target1-BsF 1 mu L, MsGA3ox1-Target2-BSR 1 mu L and template DNA 1 mu L, ddH2O7. mu.L, total volume of reaction 20. mu.L.
The reaction procedure is as follows: a first round: denaturation at 95 deg.C for 3 min; and a second round: denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1min, 35 cycles; and a third round: extension at 72 ℃ for 5 min. After the reaction is finished, the product is detected by 1% agarose gel electrophoresis, the size of the product fragment is 841bp, and if a specific band with the corresponding size exists, the sample is positive for the transgene (shown as a in FIG. 4).
2. And (3) mutation mode identification:specific primers MsGA3ox1-TF and MsGA3ox1-TR are designed in the genome sequence near target points 1 and 2 of a target gene MsGA3ox1 for amplification, and the reaction system is as follows: 2 XTaq mix 15 mu L, MsGA3ox1-TF 1 mu L, MsGA3ox1-TR 1 mu L, template DNA 1 mu L, ddH2O12. mu.L, total volume 30. mu.L.
MsGA3ox1-TF：5′-TCACTACAAGAACTTCCTGAATC-3′；
MsGA3ox1-TR：5′-ATCAAATTGGGCTTTTGAGCCAG-3′。
The reaction procedure is as follows: a first round: denaturation at 95 deg.C for 3 min; and a second round: denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1min, 35 cycles; and a third round: extension at 72 ℃ for 5 min. After the reaction, 5. mu.L of the product was detected by 1% agarose gel electrophoresis, and the size of the product fragment was about 1119bp (see b in FIG. 4).
FIG. 4 shows the result of identifying the transgene of the regenerated plant obtained by transferring alfalfa into p6401-MsgA3ox1 vector, wherein a in FIG. 4 is the result of identifying the transgene, 1 to 7 are the regenerated plants, WT is the receptor material, and negative control "-" is ddH2O and M represent the standard molecular weight of DNA, and the results show that 3, 4, 5, 6 and 7 are transgenic positive seedlings and 1 and 2 are transgenic false positive seedlings; b is the result of fragment amplification near the target, wherein 1 to 7 are regeneration plants, WT is receptor material, and negative control "-" is ddH2O and M represent the molecular weight of the DNA standard, and the results show that 1-7 have amplification bands and are basically consistent with the size of a wild type, and the sequences of the amplification bands need to be further analyzed by Sanger sequencing.
The remaining product was recovered and ligated into the pMD18T vector, and multiple single clones were picked for sequencing validation. The mutation pattern was analyzed as in FIG. 5, where the target region is in the box, the underlined letters indicate PAM sequences, ". indicates missing bases, and lowercase letters indicate inserted bases. The mutated gene sequence is as follows:
compared with wild-type alfalfa, the Msga3ox1/-8bp/-8bp/-8bp/-36bp quadruple heterozygous mutant plant has the Msga3ox1 gene in three chromosomes mutated into the Msga3ox1/-8bp gene for the Msga3ox1 coding gene, the Msga3ox1/-8bp gene is a DNA molecule obtained by deleting 8 nucleotides in total from the 350 th and 357 th positions of the Msga3ox1 coding sequence and keeping other nucleotide sequences of the sequence 2 unchanged, and the DNA molecule is named as the Msga3ox1/-8bp-L3, and the mutation causes early translation termination; the coding sequence of the Msga3ox1/-8bp-L3 gene is a DNA molecule obtained by deleting 8 nucleotides in total from the 350-th 357 position of the sequence 2 in the sequence table and keeping other nucleotide sequences of the sequence 2 unchanged, and codes a protein Msga3ox1/-8bp-L3 consisting of 147 amino acids; the Msga3ox1 gene in one chromosome is mutated into an Msga3ox1/-36bp gene, the Msga3ox1/-36bp gene is a DNA molecule obtained by deleting 36 nucleotides in total at the 320-355 th site of the Msga3ox1 coding sequence and keeping other nucleotide sequences of the sequence 2 unchanged, and the mutation causes the premature termination of translation; the coding sequence of the Msga3ox1/-36bp gene is a DNA molecule obtained by deleting 36 nucleotides in total from the 320-th and 355-th positions of the sequence 2 in the sequence table and keeping other nucleotide sequences of the sequence 2 unchanged, and codes a protein Msga3ox1/-36bp [ Msga3ox1-3 ] consisting of 265 amino acids in figure 5.
Compared with wild alfalfa, the four-equipotential heterozygous mutant plant with the gene type of Msga3ox1/-9bp/+1bp/+1bp/+1bp, for the Msga3ox1 coding gene, the Msga3ox1 gene in one chromosome is mutated into the Msga3ox1/-9bp gene, the Msga3ox1/-9bp gene is a DNA molecule obtained by deleting 9 nucleotides in total from the 351 and 359 bits of the Msga3ox1 coding sequence and keeping other nucleotide sequences of the sequence 2 unchanged, and the mutation leads to the deletion of 3 amino acids of the coding protein; the coding sequence of the Msga3ox1/-9bp gene is a DNA molecule obtained by deleting 9 nucleotides in total from the 351-359 th site of the sequence 2 in the sequence table and keeping other nucleotide sequences of the sequence 2 unchanged, and codes a protein Msga3ox1/-9bp consisting of 374 amino acids; msga3ox1 gene in three chromosomes is mutated into Msga3ox1/+1bp gene, the Msga3ox1/+1bp gene is a DNA molecule obtained by inserting a nucleotide G between the 350-351 bit of the Msga3ox1 coding sequence and keeping other nucleotide sequences of the sequence 2 unchanged, and the mutation causes translation termination; the coding sequence of Msga3ox1/+1bp gene is a DNA molecule obtained by inserting a nucleotide G between the 350-351 th sites of the sequence 2 in the sequence table and keeping the other nucleotide sequences of the sequence 2 unchanged, and codes a protein Msga3ox1/+1bp [ Msga3ox1-12 ] consisting of 150 amino acids in figure 5.
Compared with wild alfalfa, the four-equipotential heterozygous mutant plant with the gene type of Msga3ox1/+1bp/+1bp/-8bp/-7bp has the advantages that for the Msga3ox1 coding gene, the Msga3ox1 gene in two chromosomes is mutated into the Msga3ox1/+1bp gene, the Msga3ox1/+1bp gene is a DNA molecule obtained by inserting a nucleotide G between the 350-and 351-bits of the Msga3ox1 coding sequence and keeping other nucleotide sequences of the sequence 2 unchanged, and the mutation leads to premature translation termination; the coding sequence of the Msga3ox1/+1bp gene is a DNA molecule obtained by inserting a nucleotide G between the 350-351 th sites of the sequence 2 in the sequence table and keeping other nucleotide sequences of the sequence 2 unchanged, and codes a protein Msga3ox1/+1bp consisting of 150 amino acids; the Msga3ox1 gene in one chromosome is mutated into an Msga3ox1/-8bp gene, the Msga3ox1/-8bp gene is a DNA molecule obtained by deleting the 350 th and 357 th nucleotides of the Msga3ox1 coding sequence and keeping other nucleotide sequences of the sequence 2 unchanged, and the DNA molecule is named as the Msga3ox1/-8bp-L12, and the mutation causes the premature termination of translation; the coding sequence of the Msga3ox1/-8bp-L12 gene is a DNA molecule obtained by deleting 8 nucleotides in total from the 350-th 357 position of the sequence 2 in the sequence table and keeping other nucleotide sequences of the sequence 2 unchanged, and codes a protein Msga3ox1/-8bp-L12 consisting of 147 amino acids; the Msga3ox1 gene in one chromosome is mutated into Msga3ox1/-7bp gene, the Msga3ox1/-7bp gene is DNA molecule obtained by deleting 7 nucleotides in total from the 351 rd and 357 th positions of the Msga3ox1 coding sequence (sequence 2 in the sequence table) and keeping other nucleotide sequences of the sequence 2 unchanged, and the mutation causes the translation to be terminated early; the coding sequence of the Msga3ox1/-7bp gene is a DNA molecule obtained by deleting 7 nucleotides in total from the 351-357 th position of the sequence 2 in the sequence table and keeping other nucleotide sequences of the sequence 2 unchanged, and codes a protein Msga3ox1/-7bp [ Msga3ox1-15 ] consisting of 172 amino acids in figure 5.
Compared with wild-type alfalfa, the gene type Msga3ox1/-72bp/-72bp/WT/WT hybrid plant has two chromosomes with Msga3ox1 gene mutated into Msga3ox1/-72bp gene for the Msga3ox1 coding gene, and the Msga3ox1/-72bp gene is a DNA molecule obtained by deleting 72 nucleotides in total at the 351-422 bit of the Msga3ox1 coding sequence and keeping other nucleotide sequences of the sequence 2 unchanged; the coding sequence of the Msga3ox1/-72bp gene is a DNA molecule obtained by deleting 72 nucleotides in total from the 351-422 th site of the sequence 2 in the sequence table and keeping other nucleotide sequences of the sequence 2 unchanged, and codes a protein Msga3ox1/-72bp consisting of 353 amino acids; the MsGA3ox1 coding sequence was unchanged in the alfalfa genome for the other two chromosomes. It is noted that the phenotype of the heterozygous mutant is not significantly different from that of the wild type, indicating that the tetra-allelic mutation of MsGA3ox1 in alfalfa is essential for its dwarf phenotype.
3. And (3) phenotype identification:
identifying as T above0And (5) carrying out phenotype observation on the generation positive plants under the total nutrient condition. The Msga3ox1 mutant plants had more branches and reduced plant height compared to the control plants (see FIG. 6).
Wild type and Msga3ox1-3, Msga3ox1-12 and Msga3ox1-15T0The phenotype of 15 mutant plants obtained after cuttage of generation positive plants (figure 6) was tested under total nutrient conditions. Alfalfa WT, Msga3ox1 mutant material were grown under 1/2MS culture conditions for six weeks, and plant height and internode length were counted. The Msga3ox1 mutant plants (a in fig. 6 and b in fig. 6) had reduced plant height (c in fig. 6) and reduced internode length (d in fig. 6) compared to the control plants. The average plant height of the wild type is 28.9cm, the average plant height of the mutant strain is 3.0cm, and the average plant height is reduced by 89.6%; the average length of the first internodes of the wild type is 0.9cm, the average length of the first internodes of the mutant is 0.4cm, and the reduction is 55.5%; the average length of the second internodes of the wild type is 2.6cm, the average length of the second internodes of the mutant is 0.7cm, and the reduction is 73.1%; the average length of the wild type third internode is 4.6cm, the average length of the mutant third internode is 0.8cm, and the reduction is 82.6%; the average length of the fourth internode of the wild type is 5.6cm, the average length of the fourth internode of the mutant is 0.5cm, and the reduction is 91.1 percent; the average length of the wild type fifth internode is 6.1cm, the average length of the mutant fifth internode is 0.4cm, and the reduction is 93.4%; the average length of the wild type sixth internode is 5.6 cm; the average length of the wild type seventh internode is 3.6 cm; the average length of the wild type segment VIII is 1.7 cm. The mutant plant has no sixth, seventh and eighth internodes because the plant height and internode length are reduced compared with the wild type.
External source0.1mM of plant Gibberellin (GA) was applied4) The average plant height and average internode length of the Msga3ox1 mutant plants were restored to the level of wild type plants (e in FIG. 6).
Example 2 editing alfalfa MsNP1 Gene with optimized CRISPR/Cas9 System to obtain Male sterile Material
Construction of Gene editing vector p6401-MsNP1
1. Gene editing target design
The alfalfa MsNP1 contains a coding sequence (CDS) and is the MsNP1 coding gene shown in a sequence 3 in a sequence table. Predicting websites through online targets according to the genome sequence of alfalfa MsNP 1: (http://crispor.tefor.net/) Generate a list of targets, select target1 to be 5'-GCTACATTGAAGCCGCTAG-3' and target2 to be 5'-TCTCTTTGAGAACCTGTGA-3'.
Construction of sgRNA module: synthesis of primers from Beijing Liuhe Hua Dageney science and technology Co
MsNP1-Target1-BsF：5′-ATATATGGTCTCGCTTGGCTACATTGAAGCCGCTAGGTT-3', wherein the 18 th to 36 th nucleotides are designed MsNP1 target1 sequence;
MsNP1-Target1-F0：5′-GGCTACATTGAAGCCGCTAGGTTTTAGAGCTAGAAATAGC-3', wherein nucleotides 2 to 20 are a designed MsNP1 target1 sequence;
MsNP1-Target1-R0：
5′-AACTCACAGGTTCTCAAAGAGACAATTTAATGGTTCGCTTGTA-3', wherein nucleotides 4 to 22 are the reverse complement of the designed MsNP1 target 2;
MsNP1-Target-BsR：5′-ATTATTGGTCTCGAAACTCACAGGTTCTCAAAGAGAc-3', wherein the 18 th to 36 th positions are designed reverse complement sequences of MsNP1 target 2;
and amplifying by bridging PCR to obtain a sgRNA module MsNP1-5CBC with a target sequence.
Sequencing results show that the MsNP1-5CBC is shown as a sequence 5 in a sequence table. The 18 th-36 th site of the sequence 5 in the sequence table is the coding sequence of sgRNA1, and the 806 th-824 th site is the coding sequence of sgRNA 2.
3. Construction of a binary vector for gene editing: the MsNP1-5CBC fragment with the target sequence and the p6401 vector were cleaved by restriction enzyme BsaI using Golden Gate reaction and ligated by T4 DNA ligase to construct p6401-MsNP1 (see FIG. 7). After the reaction is finished, the product is added into escherichia coli TOP10 competent cells, ice bath is carried out for 30min, heat shock is carried out for 90s at 42 ℃, LB liquid culture medium without antibiotic is added, shaking culture is carried out at 37 ℃ and 150rpm for 45min, then bacterial liquid is coated on LB solid culture medium containing Kanamycin (50mg/L), and dark inversion screening culture is carried out at 37 ℃ for 12 h. Single clones are picked to 400 mu L LB liquid culture medium containing Kanamycin (50mg/L) and are shaken at 37 ℃ and 230rpm for 6-8 h, then bacterial liquid PCR identification is carried out, amplification is carried out by using the MsNP1-Target1-BsF and the MsNP1-Target2-BsR as primers, and positive clones have specific bands with the size of 841 bp. Extracting positive cloning plasmids, and determining the sequence of the constructed transformation vector p6401-MsNP1 by sequencing, wherein sequencing primers are the primers MsNP1-Target1-BsF and MsNP1-Target 2-BsR.
Sequencing results show that the p6401-MsNP1 is a recombinant expression vector obtained by replacing the fragments between BsaI restriction enzyme cleavage sites of the vector p6401 with the base sequences shown at positions 14-824 of the fragment sequence 5 and keeping the other sequences of the vector p6401 unchanged. p6401-MsNP1 expresses two sgrnas targeting sgRNA1 (target 1) and sgRNA2 (target 2) of the MsNP1 gene.
Second, agrobacterium tumefaciens mediated genetic transformation of alfalfa leaf discs
1. The gene editing binary vector p6401-MsNP1 was transferred into Agrobacterium tumefaciens EHA 105: adding 50ng of plasmid into the competent cell of Agrobacterium tumefaciens EHA105, and carrying out ice bath for 5 min; adding the mixture into a precooled electric shock cup, carrying out high-voltage electric shock at 1600V, then adding 500 mu L of YEP liquid culture medium without antibiotics, blowing and mixing uniformly, transferring the bacterial liquid into a centrifuge tube, shaking and culturing at 28 ℃ and 200rpm for 1h, taking 30 mu L of bacterial liquid, coating the bacterial liquid on YEP solid screening culture medium containing Rifampicin (75mg/L) and Kanamycin (50mg/L), carrying out dark inversion screening and culturing at 28 ℃ for 24-48 h, selecting a single clone for colony PCR identification, carrying out amplification reaction by taking MsNP1-Target1-BsF and MsNP1-Target2-BSR in the step (1) as primers, wherein the positive clone has a specific band with the size of 841bp, and selecting a positive clone (the Agrobacterium tumefaciens obtained by introducing p6401-MsNP1 into Agrobacterium tumefaciens EHA105 and is named as A105/p6401-MsNP1) for carrying out next infection.
2. Preparing an explant: taking the 5 th to 8 th fully-unfolded compound leaves of the alfalfa branch as an explant, which are taken as the receptor materials and counted from the top bud downwards, immersing the collected compound leaves in 0.1% Triton X100 aqueous solution for 5min, and washing the compound leaves for 3-5 times by using deionized water; and then transferring to a sterilized sealed bottle, adding 30% bleaching water (the effective component is 0.5% NaClO) for sterilization for 10min, and cleaning for later use in an ultra-clean workbench for 3-5 times by using the sterilized water.
3. Preparing an agrobacterium infection solution: the EHA105/p6401-MsNP1 colonies were transferred to 200mL YEP broth containing Rifamicin (75mg/L) and Kanamycin (50mg/L) and shaken at 28 ℃ and 230rpm to OD600nmIs 0.2-0.4. Transferring the EHA105/p6401-MsNP1 bacterial solution into a sterile centrifuge bottle, centrifuging at room temperature 5000rpm for 10min, removing supernatant, and resuspending with 200mL of SM4 liquid medium (containing 0.1mM of acetosyringone) to prepare an infection solution.
4. Agrobacterium-mediated genetic transformation and co-cultivation: putting the explant into the prepared staining solution, vacuumizing to-0.09 MPa for 5min, and slowly deflating; performing ultrasonic treatment for 3min at 20 ℃ in an ultrasonic cleaner; vacuumizing again to-0.09 MPa for 5min, and slowly deflating; removing the staining solution in a superclean workbench, and sucking the redundant liquid on the surface of the explant by using filter paper; the compound leaves were cut with a knife, the petioles were removed, and the cut leaves were plated on SM4 solid medium (containing acetosyringone 0.1mM) and incubated at 22 ℃ in the dark for 3 days.
5. Callus induction and differentiation: transferring the explants to SM4 solid medium (containing 200mg/L of timentin) containing the screening antibiotic hygromycin B (10mg/L) to induce resistant callus, culturing at 22 ℃, 16h of light and 8h of dark, subculturing once every 2 weeks for 6 weeks; then transferring the callus to MSBK solid medium (containing 200mg/L of timentin) containing screening antibiotic hygromycin B (10mg/L) to induce bud differentiation, culturing at 22 ℃, 16h of light and 8h of dark for 3 weeks; transferring the embryogenic callus to SH9 solid culture medium (containing 200mg/L of timentin) containing screening antibiotic hygromycin B (5mg/L) to continuously induce bud differentiation, culturing at 22 ℃ for 16h in the light and 8h in the dark, and subculturing once every 3 weeks until regeneration seedlings are generated; transferring the regenerated seedlings to 1/2MS solid culture medium without antibiotics for rooting, subculturing once every 3-4 weeks, and culturing at 22 ℃, 16h of light and 8h of dark; after rooting, transferring the seedlings into nutrient soil, covering the seedlings with a preservative film for moisture preservation and culture for 5 days, and then uncovering the film, wherein the greenhouse condition is 22 ℃, 16 hours of illumination, 8 hours of darkness and 70-80% of humidity for culture.
Third, genotype identification of alfalfa regenerated seedlings
1. And (3) transgene identification: randomly selecting 19 regenerated seedlings for genotype identification, taking a single leaf to extract genome DNA, and then carrying out PCR detection. Plasmid p6401-MsNP1 is used as a positive control, wild type genome DNA is used as a negative control, primers are MsNP1-Target1-BsF and MsNP1-Target2-BsR for amplification, and the reaction system is as follows: 2 XTaq mix 10 mu L, MsNP1-Target1-BsF 1 mu L, MsNP1-Target2-BSR 1 mu L, template DNA 1 mu L, ddH2O7. mu.L, total volume of reaction 20. mu.L.
The reaction procedure is as follows: a first round: denaturation at 95 deg.C for 3 min; and a second round: denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1min, 35 cycles; and a third round: extension at 72 ℃ for 5 min. After the reaction is finished, the product is detected by 1% agarose gel electrophoresis, the size of the product fragment is 841bp, if a specific band with corresponding size exists, the sample is positive for transgenosis, the result is shown in figure 8, wherein M represents DNA standard molecular weight, the positive control "+" represents vector p6401-MsNP1 plasmid, the negative control "-" represents receptor material, namely wild type, and serial numbers 1-19 represent genetically transformed regenerated plants, and the result shows that 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 represent positive plants for transgenosis and 1, 3, 4 represent false positive plants for transgenosis; the results showed a transgene positive rate of 84.2% (16/19).
2. And (3) mutation mode identification: specific primers MsNP1-TF and MsNP1-TR are designed in the genome sequence near target points 1 and 2 of a target gene MsNP1 for amplification, and the reaction system is as follows: 2 × Taq mix 15 μ L, MsNP1-TF 1 μ L, MsNP1-TR 1 μ L, template DNA 1 μ L, ddH2O12. mu.L, total reaction volume 30. mu.L.
MsNP1-TF：5′-TTCAAGGATGCACGAGGGAC-3′；
MsNP1-TR：5′-AGGTCATGCCATGCCATACC-3′。
The reaction procedure is as follows: a first round: denaturation at 95 deg.C for 3 min; and a second round: denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 1min, 35 cycles; and a third round: extension at 72 ℃ for 5 min. After the reaction, 5. mu.L of the product was detected by 1% agarose gel electrophoresis, and the size of the product fragment was about 486bp, as a result, see FIG. 9.
FIG. 9 shows the result of identifying the MsNP1 genotype of a regenerated plant obtained by transferring alfalfa into p6401-MsNP1 vector, wherein M represents DNA standard molecular weight, WT represents acceptor material, and serial numbers 1-19 represent genetically transformed regenerated plants, and the result shows that each sample has an amplification band.
And (3) carrying out Sanger sequencing on a part of the rest PCR products, analyzing the conditions of a peak pattern near a target point by using a sequencing primer MsNP1-TF, and primarily judging whether the editing occurs, wherein the result is shown in figure 10, wherein WT is receptor material, and 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 are all transgenic positive plants. The results show that fragments near the target point in 16 transgenic positive seedlings all show a multi-peak phenomenon, which indicates that 16 positive seedlings all undergo gene editing, namely the efficiency of obtaining gene editing plants is 100% (16/16); the remaining PCR products were recovered directly, ligated into pMD18T vector, and multiple single clones were picked for sequencing validation. The specific mutation pattern is shown in fig. 11, wherein the square box is the target region, the underlined letters are PAM sequences, "-" are missing bases, and lowercase letters are inserted bases; the result shows that the single target gene editing efficiency is 96.9%.
The plant gene editing efficiency refers to the number of plants subjected to gene editing/the number of transgenic positive plants (16/16-100%), and the single Target gene editing efficiency is calculated by the number of alleles subjected to editing at Target 1/the number of alleles in all transgenic positive plants 62/64-96.875% (specifically, by analyzing the mutation mode of the 16 transgenic positive seedlings, we find that only 1 MsNP1 wild-type copy is reserved in each of 2 plants, and the rest are edited, so that the number of the edited alleles is 4-16-2-62, and the number of all the alleles is 4-16-64).
For the genotype identification of the male sterile line obtained by mutation, mutant Mspp 1/-2bp/(-2bp/+1bp)/(-6bp/+1bp)/(-9bp/-6bp) plants (No. 5 plants in FIG. 11) are taken as an example.
Compared with wild alfalfa, the four-allelic mutant plant (the plant No. 5 in the figure 11) with the genotype of Mspp 1/-2bp/(-2bp/+1bp)/(-6 bp), for the Mspp 1 gene, the Mspp 1 gene in one chromosome is mutated into the Mspp 1/-2bp gene, and the Mspp 1/-2bp gene is a DNA molecule obtained by deleting the 1066-fold 1067-fold nucleotide GC of the sequence 3 in the sequence table and keeping other nucleotides of the sequence 3 unchanged, so that translation is terminated early; the coding sequence of the Mspp 1/-2bp gene is a DNA molecule obtained by deleting the GC nucleotide at 1066-1067 position of the sequence 3 in the sequence table and keeping other nucleotides of the sequence 3 unchanged, and codes a protein Mspp 1/-2bp consisting of 355 amino acids. The MsNP1 gene in the other chromosome is mutated into an Mspp 1/-2bp/+1bp gene, the Mspp 1/-2bp/+1bp gene is a DNA molecule obtained by deleting 1066-1067-bit nucleotide GC of a sequence 3 in a sequence table, inserting 1 nucleotide T between 1084-bit nucleotide and 1085-bit nucleotide and keeping other nucleotides of the sequence 3 unchanged, so that translation is terminated early; the coding sequence of the Mspp 1/-2bp/+1bp gene is a DNA molecule obtained by deleting the GC nucleotide at 1066-1067 position of the sequence 3 in the sequence table, inserting 1 nucleotide T between the 1084-position nucleotide and the 1085-position nucleotide and keeping other nucleotides of the sequence 3 unchanged, and codes a protein Mspp 1/-2bp/+1bp consisting of 355 amino acids. The MsNP1 gene in the other chromosome is mutated into an Mspp 1/-6bp/+1bp gene, the Mspp 1/-6bp/+1bp gene is a DNA molecule obtained by deleting the 1062-1067 th site of the sequence 3 in the sequence table, inserting 1 nucleotide T between the 1084 th and 1085 th nucleotides and keeping the other nucleotides of the sequence 3 unchanged, so that the translation is terminated early; the coding sequence of the Mspp 1/-6bp/+1bp gene is a DNA molecule obtained by deleting 6 nucleotides in total from 1062-1067 th site of the sequence 3 in the sequence table, inserting 1 nucleotide T between 1084 th and 1085 th nucleotides and keeping other nucleotides of the sequence 3 unchanged, and codes a protein Mspp 1/-6bp/+1bp consisting of 430 amino acids. The MsNP1 gene in the other chromosome is mutated into an Mspp 1/-9bp/-6bp gene, the Mspp 1/-9bp/-6bp gene is a DNA molecule obtained by deleting 9 nucleotides in total at 1063-1071 position of a sequence 3 in a sequence table, simultaneously deleting 6 nucleotides at 1084 position and 1089 position and keeping other nucleotides of the sequence 3 unchanged, and the mutation causes 5 amino acids of the encoded protein to be deleted; the coding sequence of the Mspp 1/-9bp/-6bp gene is a DNA molecule obtained by deleting 9 nucleotides in total from 1063-1071 position of the sequence 3 in the sequence table, deleting 6 nucleotides from 1084-1089 position and keeping other nucleotides of the sequence 3 unchanged, and codes a protein Mspp 1/-9bp/-6bp consisting of 575 amino acids.
Compared with wild alfalfa, the three-allelic mutant plant (14 plant in a figure 11) with the genotype of Mspp 1/-23bp/-5bp/-21bp/wt has the advantages that for the Mspp 1 gene, the Mspp 1 gene in one chromosome is mutated into the Mspp 1/-23bp gene, and the Mspp 1/-23bp gene is a DNA molecule obtained by deleting 23 nucleotides in total from the 1046-position and 1068-position of the sequence 3 in a sequence table and keeping other nucleotides of the sequence 3 unchanged, so that translation is terminated early; the coding sequence of the Mspp 1/-23bp gene is a DNA molecule obtained by deleting 23 nucleotides in total from the 1046 th and 1068 th positions of the sequence 3 in the sequence table and keeping other nucleotides of the sequence 3 unchanged, and codes a protein Mspp 1/-23bp consisting of 348 amino acids. The MsNP1 gene in the other chromosome is mutated into an Mspp 1/-5bp gene, the Mspp 1/-23bp gene is a DNA molecule obtained by deleting 5 nucleotides in total at 1063-1067 site of the sequence 3 in the sequence table and keeping other nucleotides of the sequence 3 unchanged, so that translation is terminated early; the coding sequence of the Mspp 1/-5bp gene is a DNA molecule obtained by deleting 5 nucleotides in total from 1063-1067 position of the sequence 3 in the sequence table and keeping other nucleotides of the sequence 3 unchanged, and codes a protein Mspp 1/-5bp consisting of 354 amino acids. The MsNP1 gene in the other chromosome is mutated into an Mspp 1/-21bp gene, the Mspp 1/-21bp gene is a DNA molecule obtained by deleting 28 nucleotides in total at the 1052-1079 th site of the sequence 3 in the sequence table, adding 7 nucleotides TTACCAA in a deletion section and keeping other nucleotides of the sequence 3 unchanged, and the mutation causes the encoded protein to delete 7 amino acids; the coding sequence of the Mspp 1/-21bp gene is a DNA molecule obtained by deleting 28 nucleotides in total from 1052-1079 th site of a sequence 3 in a sequence table, adding 7 nucleotides TTACCAA in a deletion section and keeping other nucleotides of the sequence 3 unchanged, and codes a protein Mspp 1/-21bp consisting of 573 amino acids. The other chromosome is not mutated; thereby producing a heterozygous form of the triallel mutant plant. It should be noted that the fertility of the Mspp 1 triallel mutant is not significantly different from that of the wild type, which indicates that the four-allelic mutation of Mspp 1 in alfalfa is essential for its male sterile phenotype.
The triallelic mutant plant can be used as a male sterility maintainer line. As shown in fig. 16, the three-allelic mutant (maintainer line) is used as a male parent to be crossed with the four-allelic mutant (male sterile line), and the progeny can be obtained according to the ratio of 1: 1 to produce a male sterile line and a maintainer line, thereby stably maintaining both materials; a four-allelic mutant (male sterile line) was used as a female parent, and an excellent pollen donor was selected for hybridization, thereby producing a hybrid F1.
3. Phenotypic identification
The phenotype identification of the obtained mutants is carried out by taking mutant Mspp 1/-2bp/(-2bp/+1bp)/(-6bp/+1bp)/(-9bp/-6bp) plants (hereinafter referred to as Mspp 1 mutants) as an example for illustration.
The inflorescences of the Mspp 1 mutant and a Control material (Control, false positive plants obtained by genetic transformation, no mutation of a target gene and consistent genotype with a receptor material) are shown in figure 12, and the results show that the inflorescences of the Mspp 1 mutant have no obvious difference in inflorescence structure and flower organ morphology compared with the wild type; the close-up of the pistils and stamens of the Mspp 1 mutant and the Control material (Control) is shown in FIG. 13, and the result shows that the pistils and the controls of the Mspp 1 mutant have no morphological difference, but the pollen of the Control material is scattered from the anther, and the stamens of the Mspp 1 mutant have no pollen exposure; the results of the amantadine staining experiments of the Msnp1 mutant and the Control material (Control) as shown in fig. 14 show that the pollen grains of the Control material are nearly spherical, can be stained to be purple red and can be released from the anther, the content of the anther of the Msnp1 mutant cannot be stained to be purple red, and no mature pollen grains exist; the Mspp 1 mutant and Control material (Control) I2-KI staining experiments are shown in FIG. 15, and the results show that the Control material pollen grains are nearly spherical and can be stained tan, while the Mspp 1 mutant anthers are non-stained pollen grains; the above results indicate that the Msnp1 tetra-allelic variant is male sterile.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Sequence listing
<110> university of agriculture in China
<120> alfalfa CRISPR/Cas9 genome editing system and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1576
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtgttagcta ttttaattga agtattatct tcctttacct cttatattga ttcagctaaa 60
cttgggttat ttgaagaagc aaatgagcta tcaatgagat ctacacttgc agagtcagtt 120
tacaatgatg ttgtttttgg aatatgccta tcttatatga tcaatgaggc atttaattgg 180
gtgcatatga tatgatggtg gaaaaaggtg ctgctcctgg cttgggaatg atgactcatg 240
tggaatttgg tcttaaattt atcacatcct tttgggatgt gatgattgta tcacttgttc 300
attttgcaaa gacaaggtgc actgctacaa actttggttt aatctgaaat aaaacaaaac 360
tcactgagag gaagatgcat cccagtaggt gaaagttgag aagtatttgc atgttactat 420
tacacttgct ttttagtccc acatcgactg aaacagaaaa tatttcagcg tttatatact 480
tcaagcgaac cagtaggctt gnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc 540
aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 600
ttttgcaaaa ttttccagat cgatttcttc ttcctctgtt cttcggcgtt caatttctgg 660
ggttttctct tcgttttctg taactgaaac ctaaaatttg acctaaaaaa aatctcaaat 720
aatatgattc agtggttttg tacttttcag ttagttgagt tttgcagttc cgatgagata 780
aaccaataga gtgtcgtttt agtaaaaaaa attattttaa aatgaatatc atcacttttc 840
aatatagaat tattatttta cttccaatta taccctctaa ttaatttcca aagcattata 900
ccaatagtaa ataaagttag tttagtaaaa ttgtcatatc ttttaacatt attattagat 960
ttcttaattt gtgtttaaaa gctttaaacg atgatcattt ttaaacagag agtataaagt 1020
agtaaaatag tactattaga aatgaattga cgtgacatgc tatgaaaagt ctggaagagt 1080
atcgataaaa ggctacacta gaggtagcta cttatatgcg caggaactga aatcaaaaat 1140
gaaataaagg agaaggaaga tgcatgttgt gttatataag tgaaggagaa ggacttgcat 1200
gttgtgttat atttgcttgt tttagtccca catcgactga aacagaaagt atctcggcgt 1260
ttatatacta caagcgaacc attaaattgn nnnnnnnnnn nnnnnnnngt tttagagcta 1320
gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg 1380
gtgctttttt ttgcaaaatt ttccagatcg atttcttctt cctctgttct tcggcgttca 1440
atttctgggg ttttctcttc gttttctgta actgaaacct aaaatttgac ctaaaaaaaa 1500
tctcaaataa tatgattcag tggttttgta cttttcagtt agttgagttt tgcagttccg 1560
atgagataaa ccaata 1576
<210> 2
<211> 1134
<212> DNA
<213> alfalfa (Medicago sativa)
<400> 2
atgccttcac tctcagaagc ctatagagct catccggtgc atgttaacca caagcaccct 60
gatttcaact cactacaaga acttcctgaa tcatacactt ggacacacct tgatgatcac 120
acccttatta attccaataa tactatgaag gagagtgcta atagtagtag tgttcccata 180
attgatctca atgacccaaa tgcttcaaag ttaataggac atgcatgcaa aacatggggt 240
gtgtatcaag tggtgaacca tggcatccca ataagcctcc ttgatgaaat tcaatggctt 300
ggacaaactc tcttcaccct tccctctcac caaaaactca aagccattcg ttccccggat 360
ggtgtttcag gttatggcct cgctcgcatc tcctccttct tccccaaact catgtggtcc 420
gagggattta ccatcgttgg atcccctctt gatcattttc gacaactttg gcctcaagat 480
tatgccaaac actgtgatac tgtcttgcaa tatgatgaag ccatgaaaaa gttagcgggg 540
aaactaatgt ggctaatgtt ggactctctt ggtattacaa tggaagatat cgaatgggct 600
ggctcaaaag cccaatttga tgagaaagcc tgtgcagcca tgcaactcaa ctcctaccct 660
agttgtccgg atccggatca cgccatgggt ctcgccccgc acacggactc aacatttcta 720
acgatccttt cccaaaatga cataagcggg ttgcaagttc aacgagaagg ttccgggtgg 780
gtcaatgtac ccccactcca tggaggactc gtggtcaacg taggcgacct attccacatt 840
ttgtcgaacg gattgtacac aagtgtgctc catcgggttt tagtgaaccg aactcgtcag 900
aggttttcgg ttgcgtattt gtatgggccc ccatcaaatg tagagatttg tccacatgag 960
aaattagtag gcccaacaca acctcccctt tataggtcag tgacttggaa tgagtacctt 1020
ggcacaaaag caaagcattt caacaaagca ctctcatccg ttagtctttg tgcacctatt 1080
aacggtttgt ttgatgtaaa cgactctaac aaaagtagtg tgcaagtggg ttaa 1134
<210> 3
<211> 1743
<212> DNA
<213> alfalfa (Medicago sativa)
<400> 3
atgagtgctt tgctgtggag gttaattctt ctttttcttg ctgggattgt tttctctcct 60
aaacattgtg tatctttaaa agatacaggt agaaaataca gttttatgca agatgcaact 120
tcagcaccaa tcatatcctt ctatgattac atcataattg gtggtggcac tgcagggtgt 180
cctttggctg caacactatc ccaaaatcat agggttttgg tgcttgaacg tggtggatca 240
ccttatggca acccaaacat aacaaattta agtgcctttg gtgttgcact ttctgataca 300
tctccttcct ctcctgctca acgattcatt tctgaagatg gtgttatcaa ctcaagggct 360
cgtgttctag gtggtggaag ttgtttgaat gctggcttct atactcgtgc aagccctcgc 420
tacgtaagtg aagctggttg ggatgaaaaa ttagtgaagg aatcatataa atgggtggag 480
agagtggtgg cattttggcc tcctatgcgt caatggcaat cagcagttag ggatggatta 540
ttggaagtag gtgtattgcc tgacaatggc tttacttatg atcatattca tgggactaag 600
gttggaggta caatctttga ccaaaatggt caaagacaca ctgctgctga tcttttggaa 660
tatgctaaca ccaacacaat tactcttctt ttgcatgcca ccgttcatag aatcttgttt 720
acaaaaagca aaggtggatt aatttcaaag ccaattgcac atggagttgt attcaaggat 780
gcacgaggga cagaacatag ggcatacttg aaacaaggga ttaggaatga gatcatagta 840
tccgctggtg cactaggtag cccacaactt ctgatgttga gtggaattgg agcagcacat 900
caccttaggg aacacaacat cagtgtagtg ttacatcaac catttgtagg acaaggaatg 960
tcagacaacc caatgaactc tgtttatgtc ccatctcctt ccccggtaga ggtttccctc 1020
atttctgttg ttggcattac caactttggc agctacattg aagccgctag cggcacaacc 1080
ttcacaggtt ctcaaagaga cttcggaatg ttttctccca agattggtca attttcaaag 1140
ttgccaccaa agcaaaggac cccagaagcc atagcaaaag caatagagag gatggagagc 1200
ttggaccaag aagctttcag gggtggattc attctagaaa aaatcttggg gccaatttca 1260
acgggtcatt tggagctccg aaacaccgat ccaaatgaga accctttggt aacatttaac 1320
tacttccaag acccaagaga cttagaaaga tgcatacaag gcatgagcac aattgagaaa 1380
attatagatt caaatgcttt ttctccattt aaatacacaa acatgtcagt ttctatgcta 1440
cttaacatga cagcaaattc accagtgaat ttattgccta aacacacaaa tacttcaatg 1500
tctttggaac aattttgtag agacactgtg atgactatat ggcattatca tggtggttgt 1560
caagttggta gggttgttga taatgattat aaggttcttg gtgtccatgc attgcgcgta 1620
atcgacggtt ctacctttaa tcattctcct ggaacaaatc ctcaagccac tgttatgatg 1680
cttggaaggt atatgggagt caaaatattg agagagagat ttgctgctga tgaaaccact 1740
taa 1743
<210> 4
<211> 841
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atatatggtc tcgcttgaca ccatccgggg aacgaagttt tagagctaga aatagcaagt 60
taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttt 120
gcaaaatttt ccagatcgat ttcttcttcc tctgttcttc ggcgttcaat ttctggggtt 180
ttctcttcgt tttctgtaac tgaaacctaa aatttgacct aaaaaaaatc tcaaataata 240
tgattcagtg gttttgtact tttcagttag ttgagttttg cagttccgat gagataaacc 300
aatagagtgt cgttttagta aaaaaaatta ttttaaaatg aatatcatca cttttcaata 360
tagaattatt attttacttc caattatacc ctctaattaa tttccaaagc attataccaa 420
tagtaaataa agttagttta gtaaaattgt catatctttt aacattatta ttagatttct 480
taatttgtgt ttaaaagctt taaacgatga tcatttttaa acagagagta taaagtagta 540
aaatagtact attagaaatg aattgacgtg acatgctatg aaaagtctgg aagagtatcg 600
ataaaaggct acactagagg tagctactta tatgcgcagg aactgaaatc aaaaatgaaa 660
taaaggagaa ggaagatgca tgttgtgtta tataagtgaa ggagaaggac ttgcatgttg 720
tgttatattt gcttgtttta gtcccacatc gactgaaaca gaaagtatct cggcgtttat 780
atactacaag cgaaccatta aattgaagcc attcgttccc cggagtttcg agaccaataa 840
t 841
<210> 5
<211> 841
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atatatggtc tcgcttggct acattgaagc cgctaggttt tagagctaga aatagcaagt 60
taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttt 120
gcaaaatttt ccagatcgat ttcttcttcc tctgttcttc ggcgttcaat ttctggggtt 180
ttctcttcgt tttctgtaac tgaaacctaa aatttgacct aaaaaaaatc tcaaataata 240
tgattcagtg gttttgtact tttcagttag ttgagttttg cagttccgat gagataaacc 300
aatagagtgt cgttttagta aaaaaaatta ttttaaaatg aatatcatca cttttcaata 360
tagaattatt attttacttc caattatacc ctctaattaa tttccaaagc attataccaa 420
tagtaaataa agttagttta gtaaaattgt catatctttt aacattatta ttagatttct 480
taatttgtgt ttaaaagctt taaacgatga tcatttttaa acagagagta taaagtagta 540
aaatagtact attagaaatg aattgacgtg acatgctatg aaaagtctgg aagagtatcg 600
ataaaaggct acactagagg tagctactta tatgcgcagg aactgaaatc aaaaatgaaa 660
taaaggagaa ggaagatgca tgttgtgtta tataagtgaa ggagaaggac ttgcatgttg 720
tgttatattt gcttgtttta gtcccacatc gactgaaaca gaaagtatct cggcgtttat 780
atactacaag cgaaccatta aattgtctct ttgagaacct gtgagtttcg agaccaataa 840
t 841
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