阅读说明：本技术 一种植物育性相关蛋白及其应用 (Plant fertility-related protein and application thereof ) 是由 蒋炳军 孙�石 陈莉 韩天富 岳岩磊 侯文胜 刘路平 袁珊 武婷婷 于 2020-01-09 设计创作，主要内容包括：本发明公开了一种植物育性相关蛋白及其应用。本发明保护通过降低或抑制目的植物中GmMS1蛋白的活性和/或含量得到雄性不育植物的方法。本发明还保护通过沉默或抑制目的植物中GmMS1基因的表达或敲除GmMS1基因得到雄性不育植物的方法。本发明对于植物育性研究及不育植物的育种有重要意义。(The invention discloses a plant fertility-related protein and application thereof. The present invention protects a method for obtaining male sterile plants by reducing or inhibiting the activity and/or content of the GmMS1 protein in a target plant. The invention also provides a method for obtaining male sterile plants by silencing or inhibiting the expression of the GmMS1 gene or knocking out the GmMS1 gene in target plants. The invention has important significance for plant fertility research and sterile plant breeding.)

1. A method of breeding male sterile plants comprising the steps of: reducing or inhibiting the activity and/or content of GmMS1 protein in a target plant to obtain a male sterile plant;

the GmMS1 protein is (A1) or (A2) or (A3) as follows:

(A1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(A2) a protein derived from soybean, having 98% or more identity to (a1), and being involved in male fertility of a plant;

(A3) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table and is related to the male fertility of plants.

2. A method of breeding male sterile plants comprising the steps of: silencing or inhibiting the expression of a GmMS1 gene in a target plant or knocking out a GmMS1 gene to obtain a male sterile plant:

the GmMS1 gene is a DNA molecule as follows:

(B1) a DNA molecule shown in a sequence 2 of a sequence table;

(B2) a DNA molecule shown in a sequence 7 of a sequence table;

(B3) a DNA molecule which hybridizes with the DNA molecule defined in (B1) or (B2) under stringent conditions and encodes a protein having the same function;

(B4) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the DNA sequence defined in (B1), (B2) or (B3) and having the same function.

3. The method of claim 2, wherein: the method is used for silencing or inhibiting the expression of the GmMS1 gene in the target plant or knocking out the GmMS1 gene, so that the expression level of the GmMS1 gene in the target plant is reduced or the function of the GmMS1 gene in the target plant is deleted for mutating the GmMS1 gene in the target plant.

4. The method of claim 3, wherein: the reduction of the expression level of the GmMS1 gene in the target plant or the functional deletion of the GmMS1 gene in the target plant is realized by mutating the GmMS1 gene in the target plant; the mutation is a deletion mutation and/or an insertion mutation and/or other mutations that can lead to a loss of gene function.

5. The method of claim 3 or 4, wherein: the reduction of the expression level of the GmMS1 gene in the genome of the target plant or the functional deletion of the GmMS1 gene in the genome of the target plant is realized by a gene editing technology.

6. The method of claim 5, wherein: the method for reducing the expression level of the GmMS1 gene in the genome of the target plant or functionally deleting the GmMS1 gene in the genome of the target plant is to make the GmMS1 gene in the target plant mutate by using CRISPR/Cas9 as shown in the following (A) or (B):

(A) inserting a base A between a 51 th base and a 52 th base from the 5' end of the sequence 2 in the sequence table;

(B) base A is inserted between 749 th base and 750 th base from 5' end of sequence 7 in the sequence table.

7. The method of claim 6, wherein: the target sequence of the CRISPR/Cas9 is shown as a sequence 6 in a sequence table.

The application of GhBZR3 protein or its related biological material in regulating plant fertility;

the relevant biological material is any one of the following (1) to (3):

(1) a coding gene of GhBZR3 protein;

(2) an agent for silencing or inhibiting the expression of (1) or knocking out (1) in a plant of interest;

(3) a substance for reducing or inhibiting the activity and/or content of the GmMS1 protein in a plant of interest;

the GmMS1 protein is (A1) or (A2) or (A3) as follows:

(A1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(A2) a protein derived from soybean, having 98% or more identity to (a1), and being involved in male fertility of a plant;

(A3) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table and is related to the male fertility of plants.

Use of (C1) or (C2) in plant breeding;

(C1) the method of any one of claims 1-7;

(C2) the GhBZR3 protein or its related biological material as claimed in claim 8.

10. The method or use according to any one of claims 1 to 9, wherein: the plant is (D1) or (D2) or (D3):

(D1) a dicot or monocot;

(D2) leguminous plants;

(D3) and (4) soybeans.

Technical Field

The invention relates to the field of soybean molecular genetic breeding, in particular to a plant fertility-related protein and application thereof.

Background

Soybean is the most important oil crop and high protein food crop in the world. In China, soybean is one of four major food crops, and plays an important role in ensuring national food safety, improving the life of people in urban and rural areas and increasing the income of farmers.

Soybeans are typical short-day crops, the suitable planting range of a single variety is 1-1.5 latitudes, and the flowering time and the mature period are advanced or delayed due to the change of the length of sunshine when the soybean is introduced in different latitudes, so that the yield is reduced and even the grains are not harvested. Meanwhile, the regional range of China is large, the ecological types are multiple, the breeding level is unbalanced, the alternating application of excellent germplasm resources is urgently needed to be enhanced, the overall level of soybean breeding is improved, and the per unit yield level of soybeans in China is improved.

Soybeans are typical self-pollinating crops, small in flowers and difficult to emasculate, and are not beneficial to the development of traditional artificial hybridization work. The method severely limits the mining and utilization of soybean germplasm resources and the polymerization and utilization of excellent gene loci. The soybean recurrent population selection technology based on the soybean male sterile mutant can effectively widen the genetic basis of soybean germplasm resources and has wide application value.

Disclosure of Invention

The invention aims to provide a plant fertility-related protein and application thereof.

In a first aspect, the present invention provides a method of breeding male sterile plants, comprising the steps of: reducing or inhibiting the activity and/or content of GmMS1 protein in a target plant to obtain a male sterile plant;

the GmMS1 protein is (A1) or (A2) or (A3) as follows:

(A1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(A2) a protein derived from soybean, having 98% or more identity to (a1), and being involved in male fertility of a plant;

(A3) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table and is related to the male fertility of plants.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In a second aspect, the present invention provides a method of cultivating a male sterile plant, comprising the steps of: silencing or inhibiting the expression of a GmMS1 gene in a target plant or knocking out a GmMS1 gene to obtain a male sterile plant:

the GmMS1 gene is a DNA molecule as follows:

(B1) a DNA molecule shown in a sequence 2 of a sequence table;

(B2) a DNA molecule shown in a sequence 7 of a sequence table;

(B3) a DNA molecule which hybridizes with the DNA molecule defined in (B1) or (B2) under stringent conditions and encodes a protein having the same function;

(B4) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the DNA sequence defined in (B1), (B2) or (B3) and having the same function.

The stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in that6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

The method is used for silencing or inhibiting the expression of the GmMS1 gene in the target plant or knocking out the GmMS1 gene, so that the expression level of the GmMS1 gene in the target plant is reduced or the function of the GmMS1 gene in the target plant is deleted for mutating the GmMS1 gene in the target plant.

The reduction of the expression level of the GmMS1 gene in the target plant or the functional deletion of the GmMS1 gene in the target plant is realized by mutating the GmMS1 gene in the target plant; the mutation is a deletion mutation and/or an insertion mutation and/or other mutations that can lead to a loss of gene function.

The reduction of the expression level of the GmMS1 gene in the genome of the target plant or the functional deletion of the GmMS1 gene in the genome of the target plant is realized by a gene editing technology.

In the embodiment of the invention, the reduction of the expression level of the GmMS1 gene in the genome of the target plant or the functional deletion of the GmMS1 gene in the genome of the target plant is realized by mutating a GmMS1 gene in the target plant by using CRISPR/Cas9 as shown in the following (A) or (B):

(A) inserting a base A between a 51 th base and a 52 th base from the 5' end of the sequence 2 in the sequence table;

(B) base A is inserted between 749 th base and 750 th base from 5' end of sequence 7 in the sequence table.

The target sequence of the CRISPR/Cas9 is shown as a sequence 6 in a sequence table.

The mutation of the GmMS1 gene in the target plant by using CRISPR/Cas9 as shown in the following (A) or (B) comprises the following steps: and (3) introducing the CRISPR/Cas9 knockout vector targeting the target sequence into a target plant to obtain a transgenic plant. The CRISPR/Cas9 vector can be a recombinant vector obtained by inserting a primer dimer into a Cas9/gRNA vector; the primer dimer is formed by annealing F2 (5'-TTGCGCCGAGGTCTAAGATACAG-3') and primer R2 (5'-AACCTGTATCTTAGACCTCGGCG-3').

Introducing the CRISPR/Cas9 knockout vector targeting the target sequence into a target plant, and specifically can be: plant cells or tissues are transformed by conventional biological methods such as Agrobacterium-mediated transformation, and the transformed plant tissues are grown into plants.

In the embodiment of the invention, the reduction of the expression level of the GmMS1 gene in the genome of the target plant or the functional deletion of the GmMS1 gene in the genome of the target plant is realized by performing the following (C) or (D) mutation on the GmMS1 gene in the target plant by using CRISPR/Cas 9:

(C) the deletion mutation from 18 th base to 23 th base of 5' end is generated in the sequence 2 of the sequence table;

(D) the sequence 7 of the sequence table is subjected to deletion mutation from 716 th base to 721 th base of the 5' end.

The target sequence of the CRISPR/Cas9 is shown as a sequence 4 in a sequence table.

The mutation of the GmMS1 gene in the target plant by using CRISPR/Cas9 comprises the following steps (C) or (D): and (3) introducing the CRISPR/Cas9 knockout vector targeting the target sequence into a target plant to obtain a transgenic plant. The CRISPR/Cas9 vector can be a recombinant vector obtained by inserting a primer dimer into a Cas9/gRNA vector; the primer dimer is formed by annealing F1 (5'-TTGGACGGGAACACCTGTGGCGG-3') and primer R1 (5'-AACCCGCCACAGGTGTTCCCGTC-3').

Introducing the CRISPR/Cas9 knockout vector targeting the target sequence into a target plant, and specifically can be: plant cells or tissues are transformed by conventional biological methods such as Agrobacterium-mediated transformation, and the transformed plant tissues are grown into plants.

In a third aspect, the invention protects the application of GhBZR3 protein or related biological materials thereof in regulating plant fertility;

the relevant biological material is any one of the following (1) to (3):

(1) a coding gene of GhBZR3 protein;

(2) an agent for silencing or inhibiting the expression of (1) or knocking out (1) in a plant of interest;

(3) a substance for reducing or inhibiting the activity and/or content of the GmMS1 protein in a plant of interest;

the GmMS1 protein was as described previously.

The encoding gene of the GhBZR3 protein (GmMS1 gene) is as described above.

The "substance for silencing or inhibiting the expression or knockout (1) of (1) in a target plant" or "substance for reducing or inhibiting the activity and/or content of the GmMS1 protein in a target plant" may be a CRISPR/Cas9 knockout vector or a recombinant bacterium containing the vector; the target sequence of the CRISPR/Cas9 knockout vector is shown as sequence 4 or sequence 6 in the sequence table. The CRISPR/Cas9 vector can be a recombinant vector obtained by inserting a primer dimer into a Cas9/gRNA vector; the primer dimer is formed by annealing F2 (5'-TTGCGCCGAGGTCTAAGATACAG-3') and primer R2 (5'-AACCTGTATCTTAGACCTCGGCG-3'). The CRISPR/Cas9 vector can be a recombinant vector obtained by inserting a primer dimer into a Cas9/gRNA vector; the primer dimer is formed by annealing F1 (5'-TTGGACGGGAACACCTGTGGCGG-3') and primer R1 (5'-AACCCGCCACAGGTGTTCCCGTC-3').

In a fourth aspect, the present invention also protects the use of (C1) or (C2) in plant breeding;

(C1) a method as described in any of the preceding paragraphs;

(C2) any of the GhBZR3 proteins or related biomaterials as described hereinbefore.

The breeding may be aimed at breeding male sterile plants.

Any one of the above plants is (D1) or (D2) or (D3):

(D1) a dicot or monocot;

(D2) leguminous plants;

(D3) and (4) soybeans.

The soybean can be soybean variety Jack.

The male sterile plant cultivated by any of the above methods also belongs to the protection scope of the invention.

The invention has important significance for plant fertility research and sterile plant breeding.

Drawings

FIG. 1 shows the sequencing results edited by the GmMS1 gene.

FIG. 2 shows the sterile phenotype of the plant edited by the GmMS1 gene.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Germination culture medium: 3.1G/L B5 medium base Salt (Gamborgs Basal Salt mix, Phototech G768), 20G/L sucrose, 1ml/L B5 medium Vitamin Solution (Gamborgs Vitamin Solution, Phototech G219), 7G/L agar, pH 5.8.

Co-culture liquid medium: 2.0G/L MS base Salt Mixture (Murashige & Skoog basic Salt mix), 3.9G/L morpholine ethanesulfonic acid (MES), 30G/L sucrose, 1ml/L B5 medium Vitamin Solution (Gamborgs Vitamin Solution, Phototech G219), 150mg/L dithiothreitol, 40mg/L acetosyringone, 2mg/L zeatin, pH 5.4.

Co-culture medium: 2.0G/L MS base salt mixture (Murashige & Skoog basic SaltMixture), 3.9G/L morpholine ethanesulfonic acid, 30G/L sucrose, 1ml/L B5 medium Vitamin Solution (Gamborgs Vitamin Solution, Phototech G219), 150mg/L dithiothreitol, 40mg/L acetosyringone, 2mg/L zeatin, 7G/L agar, pH 5.4.

Recovering the culture medium: 3.1G/L B5 Medium base Salt (Gamborgs Basal Salt mix, Phototech G768), 0.98G/L morpholine ethanesulfonic acid, 30G/L sucrose, 1ml/L B5 medium Vitamin Solution (Gamborgs vitamins Solution, Phototech G219), 150mg/L cefotaxime (cefotaxime), 450mg/L timentin, 1 mg/L6-Benzylaminopurine (6-Benzylaminopurine), 7G/L agar, pH 5.7.

Screening a culture medium: 3.1G/L B5 Medium base Salt (Gamborgs Basal Salt mix, Phototech G768), 0.98G/L morpholine ethanesulfonic acid, 30G/L sucrose, 1ml/L B5 medium Vitamin Solution (Gamborgs Vitamin Solution, Phototech G219), 150mg/L cefotaxime, 450mg/L timentin, 1 mg/L6-benzylaminopurine, 7G/L agar, 6mg/L glufosinate (glufosinate), pH 5.7.

Elongation culture medium: 4.0G/L MS base Salt Mixture (Murashige & Skoog basic Salt mix), 0.6G/L morpholine ethanesulfonic acid, 30G/L sucrose, 1ml/L B5 medium Vitamin Solution (Gamborgs vitamins Solution, Phototech G219), 150mg/L cefotaxime, 450mg/L timentin, 0.1mg/L indole acetic acid (3-Indoleacetic acid), 0.5mg/L gibberellin (Gibberellic acid), 1mg/L zeatin, 7G/L agar, 6mg/L glufosinate, pH 5.6.

Rooting culture medium: 2.0G/L MS base salt mixture (Murashige & Skoog basic SaltMixture), 0.6G/L morpholine ethanesulfonic acid, 20G/L sucrose, 1ml/L B5 medium Vitamin Solution (Gamborgs vitamins Solution, Phototech G219), 7G/L agar, 3mg/L glufosinate, pH 5.7.

Seeds of soybean variety Jack: reference documents: wei Liu, Bingjun Jiang, Liming Ma, Shouweii Zhang, Hong Zhai, Xin Xu, Wensheng Hou, Zhengjun Xia, cunxing Wu, Shi Sun, Tingning Wu, Li Chen, Tianfu Han, Functional differentiation of flowing Looking cells in a sobean, GmFT1a and GmFT2a/5a had positioning rolls in controlling flowing and measuring, New Phytology, 2018,217(3): 1335-1345; national germplasm bank preservation number: WDD01579, publicly available from the institute of crop science, academy of agricultural sciences, china.

Example 1 obtaining of Soybean Male Nuclear sterility Gene GmMS1

The soybean genome is sequenced and functionally analyzed, a soybean male nuclear sterile gene is found and named as GmMS1 gene, the CDS of the gene is shown as a sequence 2 in a sequence table, and the genome sequence is shown as a sequence 7 in the sequence table. The protein coded by the GmMS1 gene is named as GmMS1 protein and is shown as a sequence 1 in a sequence table.

Example 2 application of GmMS1 gene in regulation and control of soybean fertility

Construction of a Crisper/CAS9 Gene editing vector

1. gRNA target design and synthesis

Designing two target sequences respectively as

Target 1: GACGGGAACACCTGTGGCGGTGG；

Target 2: CGCCGAGGTCTAAGATACAGAGG。

In target 1 and target 2, the PAM sequence is underlined.

Two pairs of primers were designed according to the target sequence:

target 1 primer:

F1:5’-TTGGACGGGAACACCTGTGGCGG-3’；

R1:5’-AACCCGCCACAGGTGTTCCCGTC-3’。

target 2 primer:

F2:5’-TTGCGCCGAGGTCTAAGATACAG-3’；

R2:5’-AACCTGTATCTTAGACCTCGGCG-3’。

2. formation of primer dimer

Respectively diluting the primer F1 and the primer R1 to 10 mu M, and preparing a reaction system: f15 ul, R15 ul, H₂O15. mu.l. After mixing uniformly, reacting at 95 ℃ for 3min, then naturally cooling to 25 ℃, and then cooling to 16 ℃ for 5min to obtain the primer dimer 1.

Respectively diluting the primer F2 and the primer R2 to 10 mu M, and preparing a reaction system: f25 ul, R25 ul, H₂O15. mu.l. After mixing uniformly, reacting at 95 ℃ for 3min, then naturally cooling to 25 ℃, and then cooling to 16 ℃ for 5min to obtain the primer dimer 2.

3. Taking the primer dimer 1 obtained in the step 2 to prepare a reaction system: cas9/gRNA vector 1. mu.l, primer dimer 11. mu.l, Solution 11. mu.l, Solution 21. mu.l, H₂O6. mu.l. The reaction was carried out at 16 ℃ for 2 hours.

The above carriers and reagents are available from Beijing Weishanglide Biotech Co., Ltd, cat # VK 005-15.

And (3) after the reaction is finished, obtaining a recombinant vector CRISPR/Cas9-GmMS1-1, wherein the recombinant vector CRISPR/Cas9-GmMS1-1 contains a DNA molecule (sequencing verification) shown in a sequence 3 of a sequence table, and the sgRNA is expressed. The target sequence of the sgRNA is sequence 4.

4. Taking the primer dimer 2 obtained in the step 2 to prepare a reaction system: cas9/gRNA vector 1. mu.l, primer dimer 21. mu.l, Solution 11. mu.l, Solution 21. mu.l, H₂O6. mu.l. The reaction was carried out at 16 ℃ for 2 hours.

The above carriers and reagents are available from Beijing Weishanglide Biotech Co., Ltd, cat # VK 005-15.

And (3) obtaining a recombinant vector CRISPR/Cas9-GmMS1-2 after the reaction is finished, and expressing a DNA molecule containing a sequence 5 in a sequence table in the recombinant vector CRISPR/Cas9-GmMS1-2 to express sgRNA (sequencing verification is carried out). The target sequence of the sgRNA is sequence 6.

Preparation of recombinant Agrobacterium

And (3) respectively introducing the recombinant vector CRISPR/Cas9-GmMS1-1 and the recombinant vector CRISPR/Cas9-GmMS1-2 constructed in the steps (3) and (4) into the Agrobacterium tumefaciens EHA105 to obtain the recombinant Agrobacterium tumefaciens GmMS1-1 and the recombinant Agrobacterium tumefaciens GmMS 1-2.

Third, obtaining of Gene-edited Soybean

And (3) transforming the soybean by using the recombinant agrobacterium obtained in the step two and adopting an agrobacterium tumefaciens mediated soybean cotyledonary node transformation method, wherein the specific transformation method is as follows:

(1) the method comprises the steps of selecting full soybean variety Jack seeds with uniform size, no disease spots, no cracks, smooth surfaces and no wrinkles, and putting the seeds into a glass culture dish. Then put into a desiccator, and the culture dish was opened. A glass beaker was placed in the desiccator, and 100mL of sodium hypochlorite was added first, followed by dropwise addition of 4mL of concentrated hydrochloric acid to the beaker. The vaseline is coated on the periphery of the dryer cover, and then the dryer is covered to form a sealed state. The desiccator was then placed in a fume hood and the seeds were sterilized for 16-20 h.

(2) After the step (1) is finished, the sterilized seed hypocotyl is vertically and upwards inoculated into a germination culture medium, a culture dish is not sealed, and the seed hypocotyl is placed in a tissue culture room with the temperature of 25 ℃ and the illumination of 16 h/the darkness of 18 h for culture for 1 d.

(3) And (3) after the step (2) is completed, taking cotyledonary nodes of the germinated soybean seeds as explants. Firstly, peeling soybean seed coats, longitudinally cutting and separating two cotyledons, cutting a strip wound at the joint part (cotyledon node) of the cotyledons and an embryonic axis, putting the scratched explant into a co-culture liquid culture medium (bacterial liquid OD value is 0.6-0.8) containing recombinant agrobacterium heavy suspension, dip-dyeing for 30min at 28 ℃, after infection, downwards inoculating the explant (cotyledon) into the co-culture medium with the surface paved with filter paper, and culturing for 5 days under the conditions of 25 ℃, 16h illumination/18 h darkness.

(4) After completing step (3), the explants were transferred to recovery medium and cultured for 7d at 25 ℃ under 16h light/18 h dark conditions.

(5) After the step (4) is completed, the main bud generated by the explant is cut off and transferred into a screening culture medium, and the explant is cultured for 21d under the conditions of 25 ℃ and 16h of light/18 h of dark.

(6) After the step (5) is completed, the browned leaves are peeled off, and the produced adventitious bud is transferred into an elongation medium for elongation. And 4, subculturing once for 15d, and subculturing 2-3 times. During the period, the generated elongation seedlings are transferred into a rooting culture medium for rooting.

(7) And (6) finishing the step (6), taking out the seedlings from the culture medium after the roots grow out, and transplanting the seedlings into a small pot filled with the matrix for hardening the seedlings. After hardening off for 1 week, transferring the seedlings into a big pot for growth to obtain T0 generation plants.

(8) Detecting T0 generation plants by the following method:

and respectively extracting genome DNA of the transgenic plant and the wild plant, and performing PCR amplification by using the extracted genome DNA as a template and a detection primer.

F-614:CGCCATAGTGAAGTAGCGGA；

R-614:CAGTTGAAAACAAACTTACCGAAGG。

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; then 35 cycles of 95 ℃ 30sec, 56 ℃ 30sec, 72 ℃ 1 min; extension was then carried out at 72 ℃ for 10 min. The PCR product is sent to sequencing and the gene editing plant is screened.

Sequencing results show that 60T 0 generation plants are obtained after soybean is transformed by the recombinant agrobacterium GmMS1-1, 1 plant which is mutated in a GmMS1 gene and homozygous mutated is obtained in a T1 descendant, and compared with a wild type, the homozygous mutated plant has the difference that: there is a deletion of 6bp between positions 18 and 23 of sequence 2 (GGCGGT). After soybean is transformed by the recombinant agrobacterium GmMS1-2, 56 strains of T0 generation plants are obtained in total, 5 strains of T1 generation plants are plants which are mutated in a GmMS1 gene and are homozygous mutation, and compared with wild type plants, the homozygous mutation plants are different in that: there is a difference of one nucleotide (i.e., an insertion mutation occurs and is homozygous, the insertion is between the 51 st and 52 nd positions of the sequence 2, the single base of the insertion is A, the sequencing result is shown in figure 1), and the difference of the nucleotide causes frame shift, so that the GmMS1 protein can not be effectively expressed (figure 1).

Fourth, soybean fertility detection

And (3) the plant to be detected: wild type plants and 5 homozygous mutant plants (insertion mutant plants obtained by transferring recombinant agrobacterium GmMS 1-2).

And planting the plant to be tested in a pot under the outdoor natural condition.

The results are shown in FIG. 2. The result shows that the wild control plant is normal in pod bearing and full in seed, while the homozygous plant with the frame shift mutation edited by the GmMS1 gene is a male sterile plant and cannot normally bear pods. These results indicate that editing of the GmMS1 gene can lead to male sterility.

Sequence listing

<110> institute of crop science of Chinese academy of agricultural sciences

<120> a plant fertility-associated protein and uses thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 950

<212> PRT

<213> Soybean (Glycine max (Linn.) Merr.)

<400> 1

Met Thr Gly Thr Pro Val Ala Val Ala Ala Ala Thr Pro Arg Ser Lys

1 5 10 15

Ile Gln Arg Asn Ala Ser Gly Thr Pro Gly Gly Pro Lys Val Arg Glu

20 25 30

Glu Lys Ile Arg Val Thr Val Arg Met Arg Pro Leu Asn Thr Lys Glu

35 40 45

Gln Ala Met Tyr Asp Leu Ile Ala Trp Asp Cys Leu Asp Glu His Thr

50 55 60

Ile Val Phe Lys Asn Pro Asn Gln Glu Arg Pro Thr Thr Pro Tyr Thr

65 70 75 80

Phe Asp Lys Val Phe Ala Pro Thr Cys Ser Thr His Lys Val Tyr Glu

85 90 95

Glu Gly Ala Lys Asp Val Ala Leu Ser Ala Leu Ser Gly Ile Asn Ala

100 105 110

Thr Ile Phe Ala Tyr Gly Gln Thr Ser Ser Gly Lys Thr Phe Thr Met

115 120 125

Arg Gly Val Thr Glu Ser Ala Ile Lys Asp Ile Tyr Asp Tyr Ile Lys

130 135 140

Asn Thr Pro Glu Arg Asp Phe Ile Leu Arg Ile Ser Ala Leu Glu Ile

145 150 155 160

Tyr Asn Glu Thr Val Ile Asp Leu Leu Lys Arg Glu Ser Gly Pro Leu

165 170 175

Arg Leu Leu Asp Asp Pro Glu Lys Gly Thr Ile Val Glu Lys Leu Asn

180 185 190

Glu Glu Val Ala Glu Asp Arg Gln His Leu Arg Arg Leu Ile Gly Ile

195 200 205

Cys Glu Ala Gln Arg Gln Val Gly Glu Thr Ala Leu Asn Asp Lys Ser

210 215 220

Ser Arg Ser His Gln Ile Ile Arg Leu Thr Val Glu Ser Ser Leu Arg

225 230 235 240

Glu Ser Ser Gly His Val Lys Ser Tyr Ile Ala Ser Leu Asn Phe Val

245 250 255

Asp Leu Ala Gly Ser Glu Arg Ile Ser Gln Thr Asn Thr Cys Gly Ala

260 265 270

Arg Met Lys Glu Gly Ser His Ile Asn Arg Ser Leu Leu Thr Leu Ala

275 280 285

Ser Val Ile Arg Lys Leu Ser Gly Gly Lys Cys Gly His Ile Pro Tyr

290 295 300

Arg Asp Ser Lys Leu Thr Arg Ile Leu Gln Ser Ser Leu Gly Gly Asn

305 310 315 320

Ala Arg Thr Ala Ile Ile Cys Thr Ile Ser Pro Ser Leu Ser His Val

325 330 335

Glu Gln Thr Arg Asn Thr Leu Ala Phe Ala Thr Ser Ala Lys Glu Val

340 345 350

Ile Asn Thr Ala Arg Val Asn Met Val Val Ser Asn Lys Thr Leu Val

355 360 365

Arg Gln Leu Gln Lys Glu Val Ala Arg Leu Glu Gly Glu Leu Arg Ser

370 375 380

Pro Asp Leu Ser Val Asn Ser Cys Leu Arg Ser Leu Leu Ala Glu Lys

385 390 395 400

Glu Leu Lys Ile Gln Gln Met Glu Arg Asp Met Glu Asp Leu Arg Arg

405 410 415

Gln Arg Asp Leu Ala Gln Thr Gln Leu Asp Leu Glu Arg Arg Val Asn

420 425 430

Lys Val Pro Lys Gly Ser Asn Asp Cys Gly Pro Ser Ser Gln Ile Val

435 440 445

Arg Cys Leu Ser Phe Pro Glu Glu Asn Lys Ser Ala Asn Gly Lys Arg

450 455 460

Thr Pro Glu Arg Arg Glu Ala Val Gly Arg Gln Ala Met Leu Lys Asn

465 470 475 480

Leu Leu Ala Ser Pro Asp Pro Ser Ile Leu Val Gly Glu Ile Arg Lys

485 490 495

Leu Glu Asp Arg Gln Leu Gln Leu Cys Glu Asp Ala Asn Arg Ala Leu

500 505 510

Glu Val Leu His Gln Asp Phe Ala Thr His Lys Leu Gly Asn Gln Glu

515 520 525

Thr Ala Glu Thr Met Ser Lys Val Leu Ser Glu Ile Lys Asp Leu Val

530 535 540

Ala Ala Ser Ser Thr Pro Glu Glu Ile Val Ala Ala Asp Lys Ala Asp

545 550 555 560

Leu Met Glu Lys Ile Thr Gln Leu Lys Asn Gln Gly Asn Thr Ile Ala

565 570 575

Ser Leu Glu Arg Lys Leu Glu Asn Val Gln Lys Ser Ile Asp Lys Leu

580 585 590

Val Ser Ala Phe Asn Ala Glu Glu Thr Pro Glu Asn Lys Thr Thr Pro

595 600 605

Leu Arg Arg Lys Lys Ile Leu Pro Phe Thr Leu Ser Asn Ser Pro Asn

610 615 620

Met Gln His Ile Ile Arg Ala Pro Cys Ser Pro Leu Ser Ser Ser Arg

625 630 635 640

Lys Ala Met Glu His Asp Ile Glu Asn Arg Ala Pro Glu Asn Asn Ile

645 650 655

Gly Ile Ser Gly Ser Asp Ser Phe Ala Lys Phe His Lys Asp Thr Pro

660 665 670

Arg Lys Asp Asp Lys Ser Cys Asp Ser Ile Leu Ser Arg Ala Gly Ser

675 680 685

Pro Ala Thr Arg Lys Ser Lys Ser Val Asn Val Met Lys Ile Gln Lys

690 695 700

Met Phe Lys Asn Ala Ala Glu Glu Asn Ile Arg Ser Phe Arg Val Tyr

705 710 715 720

Val Thr Glu Leu Lys Glu Leu Val Ala Lys Leu His Tyr Gln Lys Gln

725 730 735

Leu Leu Val Cys Gln Val Leu Glu Leu Glu Ala Asn Lys Ser Leu Asn

740 745 750

Glu Glu Lys Asp Thr Pro Asp Arg Ser Pro Leu Pro Trp His Ile Leu

755 760 765

Phe Asp Gln Gln Arg Lys Gln Ile Ile Met Leu Trp His Leu Cys His

770 775 780

Ile Ser Leu Val His Arg Thr Gln Phe Phe Leu Leu Leu Gly Gly Asp

785 790 795 800

Pro Ser Asp Gln Ile Tyr Met Glu Val Glu Leu Arg Arg Leu Thr Arg

805 810 815

Leu Glu Gln His Leu Ala Glu Leu Gly Asn Ala Ser Pro Ala Leu Leu

820 825 830

Gly Asp Glu Pro Ala Gly Ser Val Ser Ala Ser Ile Arg Ala Leu Lys

835 840 845

Gln Glu Arg Glu His Leu Ala Arg Lys Val Asn Thr Lys Leu Thr Ala

850 855 860

Glu Glu Arg Glu Leu Leu Tyr Ala Lys Trp Glu Val Pro Pro Val Gly

865 870 875 880

Lys Gln Arg Arg Leu Gln Phe Val Asn Lys Leu Trp Thr Asp Pro Tyr

885 890 895

Asn Met Gln His Val Gln Glu Ser Ala Glu Ile Val Ala Lys Leu Ile

900 905 910

Asp Phe Ser Val Ser Asp Glu Asn Ser Lys Asp Met Ile Glu Leu Asn

915 920 925

Phe Ser Ser Pro Phe Asn Lys Lys Thr Trp Ala Gly Trp Asn Phe Ile

930 935 940

Ser Asn Leu Leu Asn Leu

945 950

<210> 2

<211> 2850

<212> DNA

<213> Soybean (Glycine max (Linn.) Merr.)

<400> 2

atgacgggaa cacctgtggc ggtggctgcg gcaacgccga ggtctaagat acagaggaat 60

gcttcaggta cgccgggtgg ccccaaagtt cgggaggaga aaattcgagt cacggttcgg 120

atgaggccgc tcaatacaaa ggagcaagct atgtacgatc taattgcttg ggattgtttg 180

gatgaacaca ctattgtgtt caagaatcca aaccaagaga ggcctacaac accatacacc 240

ttcgataaag tttttgcacc tacgtgctca actcataagg tttatgaaga aggggctaaa 300

gatgttgctt tatcagcact ttctggaatc aatgcaacaa tatttgcgta tgggcagact 360

agcagtggta agacattcac gatgagaggc gtcactgaaa gtgctattaa agacatctac 420

gactacatta agaatacacc agaaagggat tttattctga gaatctctgc tctggaaatc 480

tataatgaga ctgtcataga ccttctgaaa cgtgaatctg gtcctcttcg gctcttggat 540

gatcctgaga aagggactat tgtggaaaag ctgaatgaag aagtagctga agatcgtcaa 600

catcttaggc gcttaattgg catctgcgaa gctcaaaggc aagtgggaga aactgcttta 660

aatgataaaa gctcaagatc acatcaaata atcaggctga ctgtagaaag cagccttcgt 720

gaaagttcag gtcacgtaaa gtcttacata gcaagtttga attttgtgga tcttgctgga 780

agtgaacgca tctctcaaac aaatacatgt ggagcaagaa tgaaggaagg cagccacatc 840

aaccgaagtt tgttgacact tgcatcagtc atcaggaagc taagtggcgg aaaatgtggt 900

cacataccat atagagactc aaaattgaca cgaatattgc agtcttcatt aggagggaat 960

gctcgaacag cgattatctg taccataagt ccttccttaa gtcatgtgga gcaaacaaga 1020

aatacactag catttgctac cagtgcaaag gaagtcatta atactgcccg agttaatatg 1080

gtcgtttcaa ataagacact agttagacag ttgcaaaagg aagttgcgag gcttgaaggg 1140

gagttacgaa gccctgacct ttctgtgaat tcatgtctaa ggtcattgct agctgaaaag 1200

gagttgaaaa ttcagcagat ggagagggat atggaagatc tgaggcgaca gagagacctt 1260

gcacaaactc aacttgatct ggaaagaaga gtgaataaag ttccaaaggg atcaaacgat 1320

tgtgggccct ctagtcaaat agtcagatgt ctttcttttc ctgaagagaa caaatcagct 1380

aatggtaaac gtacgccaga gcgacgagag gcagtgggca ggcaggcaat gctgaagaat 1440

ttattggctt ctcctgatcc atccatactg gttggtgaaa tccgaaagct tgaggatcgg 1500

cagctccagc tctgtgagga tgcaaatcga gctcttgaag ttctgcacca ggattttgca 1560

actcacaaac ttgggaatca agaaactgct gaaaccatgt cgaaagtact atctgaaata 1620

aaagacttag tagctgccag ctctactcca gaagaaattg tggcagcaga taaggccgac 1680

ctaatggaaa agatcacaca gttgaaaaat caagggaaca ccattgcatc tttagaaagg 1740

aagctggaga atgttcaaaa atctatagac aagcttgtgt ctgcttttaa tgcagaggag 1800

actccagaaa acaagacgac ccctctgaga aggaagaaaa ttcttccttt cacattaagc 1860

aacagtccca acatgcagca tataatacgt gctccttgct cgcctctctc ctcttcgcgt 1920

aaagcaatgg aacatgacat tgagaacagg gcaccggaaa acaacattgg catctctggc 1980

agtgattctt ttgctaagtt tcataaagat actccacgaa aggatgataa aagttgtgat 2040

tctattttat cacgggcagg aagcccagct acaaggaaat caaaatcagt gaatgtgatg 2100

aagattcaaa agatgttcaa gaatgctgcg gaggagaaca ttcggagctt cagagtttat 2160

gttaccgagt taaaagagct agtggcaaaa ctgcattacc agaagcagct actggtttgc 2220

caggttttgg aactggaagc aaacaagtca ttaaatgaag aaaaggatac acctgatcgg 2280

tctcccttgc catggcatat actatttgat cagcagagaa agcaaattat catgttatgg 2340

catttatgcc acatatctct tgtgcaccgg acacagtttt ttcttctgtt aggaggagac 2400

ccttctgatc agatatatat ggaagttgaa cttagaagat tgactcggtt agaacagcac 2460

ctggcagagc ttgggaatgc tagtcctgca cttctaggtg atgagcctgc aggctctgtt 2520

tcagcaagca ttagagctct gaagcaagaa agggaacatc ttgctaggaa ggtgaacact 2580

aaacttacag cagaggagag ggaactgctt tatgcaaaat gggaagttcc tccagttgga 2640

aaacaaagga gactgcaatt tgtaaataaa ttgtggaccg acccttataa catgcaacat 2700

gtgcaagaaa gtgctgaaat tgtagcaaag ctcattgatt tcagtgtatc tgatgaaaac 2760

agcaaggata tgattgaatt aaacttttca agccctttta ataagaaaac atgggcgggc 2820

tggaacttta tatcaaatct tctaaatttg 2850

<210> 3

<211> 548

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttcgttgaac aacggaaact cgacttgcct tccgcacaat acatcatttc ttcttagctt 60

tttttcttct tcttcgttca tacagttttt ttttgtttat cagcttacat tttcttgaac 120

cgtagctttc gttttcttct ttttaacttt ccattcggag tttttgtatc ttgtttcata 180

gtttgtccca ggattagaat gattaggcat cgaaccttca agaatttgat tgaataaaac 240

atcttcattc ttaagatatg aagataatct tcaaaaggcc cctgggaatc tgaaagaaga 300

gaagcaggcc catttatatg ggaaagaaca atagtatttc ttatataggc ccatttaagt 360

tgaaaacaat cttcaaaagt cccacatcgc ttagataaga aaacgaagct gagtttatat 420

acagctagag tcgaagtagt gattggacgg gaacacctgt ggcgggtttt agagctagaa 480

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 540

cttttttt 548

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gacgggaaca cctgtggcgg 20

<210> 5

<211> 548

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ttcgttgaac aacggaaact cgacttgcct tccgcacaat acatcatttc ttcttagctt 60

tttttcttct tcttcgttca tacagttttt ttttgtttat cagcttacat tttcttgaac 120

cgtagctttc gttttcttct ttttaacttt ccattcggag tttttgtatc ttgtttcata 180

gtttgtccca ggattagaat gattaggcat cgaaccttca agaatttgat tgaataaaac 240

atcttcattc ttaagatatg aagataatct tcaaaaggcc cctgggaatc tgaaagaaga 300

gaagcaggcc catttatatg ggaaagaaca atagtatttc ttatataggc ccatttaagt 360

tgaaaacaat cttcaaaagt cccacatcgc ttagataaga aaacgaagct gagtttatat 420

acagctagag tcgaagtagt gattgcgccg aggtctaaga tacaggtttt agagctagaa 480

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 540

cttttttt 548

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgccgaggtc taagatacag 20

<210> 7

<211> 6884

<212> DNA

<213> Soybean (Glycine max (Linn.) Merr.)

<400> 7

ttgaagtagc cgttactata cacacctctt actatctctg gaactttgaa tttcagagcg 60

tttttttctc tttcgaaaag taaacctcgt cgtcgttcat tcgttctttc tctctttcag 120

atcgattgca gaatttattt cgccactcca aactacgtcg ttcagcattt ccaccggcga 180

ctgagcctcc tcttcttcat ctgcaagtac ttcttttctt tcatttcaca aagaatcaat 240

ttctttttca ataaacgtgc tcgattagtt gaattgaatg tgcctagtaa agatcaacaa 300

gaagaagaag aagatttagc aaaatatcat atgaaacaga tgtcaatcgc catagtgaag 360

tagcggatta ctattttgtt tttttttcgg aggtgttctg ataggtgatt tgttgggcga 420

ttcagcaatt ttaaaagctt cagatcgtgc taggttttag cttaatcttc attcgctctt 480

tttgcaatgt agctaattca gaaatcgata tccgtttttc tttctattac cgttttcatc 540

caaatcagtg acctccaaag ttgcaacggt tttcggaaga acatggctta gctattattt 600

ataatgttct ttatttcaag tattttctct ttagaaatga agtgctgtaa actgctgttg 660

ctgacggatc gatctgcaga aattgaaggc agagctcgat gacgggaaca cctgtggcgg 720

tggctgcggc aacgccgagg tctaagatac agaggaatgc ttcaggtacg ccgggtggcc 780

ccaaagttcg ggaggagaaa attcgagtca cggttcggat gaggccgctc aatacaaagg 840

agcaagctat gtacgatcta attgcttggg attgtttgga tgaacacact attgtgttca 900

agaatccaaa ccaagagagg cctacaacac catacacctt cggtaagttt gttttcaact 960

gtgttttttt tttccgagag gaatttgata aacttacatt ttcgtgaaca gaaattcatg 1020

aatttatatt taaatttctg tataaatggg aggtaatgaa tataaacctg gttaacgttc 1080

tagtgcgttt attgtttaac gttgtttgag tggatttgta tttgttaagt ttctatttcc 1140

ttttatataa catttatctt atttgttatt tgttacttgt tacctttttt cttattatta 1200

tttgttatgt tgcagataaa gtttttgcac ctacgtgctc aactcataag gtttatgaag 1260

aaggggctaa agatgttgct ttatcagcac tttctggaat caatggtaac ttgttctcag 1320

ctaatgcttg tgttagatta cgggatgttt tagatgcatt caaaagtttc atttttttgc 1380

agcaacaata tttgcgtatg ggcagactag cagtggtaag acattcacga tgagaggcgt 1440

cactgaaagt gctattaaag acatctacga ctacattaag aatgtgagtt actagtttac 1500

tgttgatacc taccaaactt tatatgtcaa ttatcttcta ctgagtataa agtagaaggc 1560

tattcatgag ttgaatccta ttagtattta tcaagattaa ttgcaggatt tgatggcaat 1620

tacttgcaat tatgaacatt ttttattcgt ttcaatttcc acagacacca gaaagggatt 1680

ttattctgag aatctctgct ctggaaatct ataatgagac tgtcatagac cttctgaaac 1740

gtgaatctgg tcctcttcgg ctcttggatg atcctgaggt atagttcagg gctaaaccct 1800

gaacggaaat ttgtcaggga tgctttaaaa tttccttgat ttgtcgcaaa tcctgaggta 1860

gattttttct ttggtacaga aagggactat tgtggaaaag ctgaatgaag aagtagctga 1920

agatcgtcaa catcttaggc gcttaattgg catctgcgaa ggtaatctag aaatctggtg 1980

tggtttgtca tgtgagacat atataggttt ctcaagagca tacagtatca ccatgttgtt 2040

tggctaagac ttttatatct cactattggt gatgttctct ttgtatagaa accatgaaaa 2100

tatataagtt gtgggcattt tgcacttagg ttatctgcat agtactattg ttcatgtaaa 2160

tattgtatct gcagctcaaa ggcaagtggg agaaactgct ttaaatgata aaagctcaag 2220

atcacatcaa ataatcaggc tggtaagctc agctgaggaa gtacaagtat tcattataat 2280

gcatgcatat ttcagattgg ttaatttacc catttatttt ctctggctac tttatgcgga 2340

gattagaaat aagacttatg cttactgctc tgtaatcact attttcatac gaatatttca 2400

gactgtagaa agcagccttc gtgaaagttc aggtcacgta aagtcttaca tagcaagttt 2460

ggtatgtttc gtcttcatag cttgacttga tagtcttgat aacaagctag tcatagtgat 2520

gaatcattat tataacctca cagaattttg tggatcttgc tggaagtgaa cgcatctctc 2580

aaacaaatac atgtggagca agaatgaagg aaggcagcca catcaaccga agtttgttga 2640

cacttgcatc agtcatcagg aagctaaggt ccaacatctt taaatatcaa aatgcatgtg 2700

gctactttgt tattgttctt ctacaaaaga gccatgatta taaaaattta gcagccaaga 2760

tcaacaagat gtagagaaaa aaaaatacta aaaggcccat ttgtttgtgt gttttttttt 2820

ataagaaaaa catttttttt aaattaaaaa tatgtttaaa atctccacaa taattgattt 2880

ttttttaaat aaaaaatcct caacaagatt ttaatatttt ttgcttttaa tcatcaaatg 2940

tcacattaaa gttgtcttgt gacatgaagg taggttcaaa tctttgaaac agtcgctcta 3000

cttacagggg gataagactg cttacatcta tcctttcaat atctcactag gaagaagcct 3060

catgcaccag gctactcttt ttgttcaaac tgaaaagttt ttgacaacta ctaatatact 3120

tgtggaatcc agtttttctc gtaatcattt tcatttgatg actagaaatt gtttactgag 3180

gaagttggtt aatttgaaat tgtttattgt ggaagttggt taatttgaaa ttggcattga 3240

ttattattat gatgaagtaa agacctgtag agctatgtaa tttggtcaat agacaggcgt 3300

ccgattcagc ttttggaggt tttcttttgc tttgcagtgg cggaaaatgt ggtcacatac 3360

catatagaga ctcaaaattg acacgaatat tgcagtcttc attaggaggg aatgctcgaa 3420

cagcgattat ctgtaccata agtccttcct taagtcatgt ggagcaaaca agaaatacac 3480

tagcatttgc taccagtgca aaggaagtca ttaatactgc ccgagttaat atggtatgat 3540

taaatgtcta aatataatta tgatttggtt gattgaagag tagattagta tgaaaatctt 3600

ttctcctcgt gttcttatat tacttgataa aatcattttt tcccaaaaat ttgttttcaa 3660

acagaagctt gcagcagttt ggcatctttc aatctttttg acaagtgtaa atttggatag 3720

tagctctcct aggtttctga tacttagttc atagcctagc ccaccttgac tagcagcaaa 3780

gtgtatttgt ttggtagaag tatttgaaat tattgagaaa agtgatggat gcaccttctt 3840

cataaaaagc accttgtctt ttatttcggg tggcaattat agaggacttg ttttcccctc 3900

tactcgctaa gtgatattca tttttttttg ttatttcgag aagccacctc attgattaca 3960

agctttcata ttttccatga aaaaagaaag gtatatacta agatagatgg tgatgatcat 4020

ttaacaggtc gtttcaaata agacactagt tagacagttg caaaaggaag ttgcgaggct 4080

tgaaggggag ttacgaagcc ctgacctttc tgtgaattca tgtctaaggt cattgctagc 4140

tgaaaaggag ttgaaaattc agcaggtaca aacttcttta cttggtctta actttcatgt 4200

taactttact gtgttaaaat ttttgaacta acctcttctc tgagtcctct agatggatat 4260

tataatggta ttctttggag ttcttacatt attattatct cttgtctcaa gatgtggtca 4320

ataatttgat ttctatgtag ctaaacattc caacattcaa gtaaaatatt catatactct 4380

ttgtgtatgc aatgggttta actttcccat tccacaatgg atcaaaatcc cgggaactgg 4440

cttcccgttg tctgcttttc ttgtcgaatg aactgtgaaa gaccaattta ttagtaacac 4500

ttaattgtct tttcttgagc ttttagaaga gaaactcttt tggatgaatt gaattgataa 4560

acacaaattt gcagatggag agggatatgg aagatctgag gcgacagaga gaccttgcac 4620

aaactcaact tgatctggaa agaagagtga ataaagttcc aaaggtattt ggaatggtca 4680

tatcacatta ttgtaaccct gatttagact tctgactcaa attatataat tcttgcaggg 4740

atcaaacgat tgtgggccct ctagtcaaat agtcagatgt ctttcttttc ctgaagagaa 4800

caaatcagct aatggtaaac gtacgccaga gcgacgagag gcagtgggca ggcaggcaat 4860

gctgaagaat ttattggctt ctcctgatcc atccatactg gttggtgaaa tccgaaagct 4920

tgaggatcgg cagctccagc tctgtgagga tgcaaatcga gctcttgaag ttctgcacca 4980

ggattttgca actcacaaac ttgggaatca agaaactgct gaaaccatgt cgaaagtact 5040

atctgaaata aaagacttag tagctgccag ctctactcca gaagaaattg tggcagcaga 5100

taaggccgac ctaatggaaa agatcacaca gttgaaaaat caagggaaca ccattgcatc 5160

tttagaaagg aagctggaga atgttcaaaa atctatagac aagcttgtgt ctgcttttaa 5220

tgcagaggag actccagaaa acaagacgac ccctctgaga aggaagaaaa ttcttccttt 5280

cacattaagc aacagtccca acatgcagca tataatacgt gctccttgct cgcctctctc 5340

ctcttcgcgt aaagcaatgg aacatgacat tgagaacagg gcaccggaaa acaacattgg 5400

catctctggc agtgattctt ttgctaagtt tcataaagat actccacgaa aggatgataa 5460

aagttgtgat tctattttat cacgggcagg aagcccagct acaaggaaat caaaatcagt 5520

gaatgtgatg aagattcaaa agatgttcaa gaatgctgcg gaggagaaca ttcggagctt 5580

cagagtttat gttaccgagt taaaagagct agtggcaaaa ctgcattacc agaagcagct 5640

actggtttgc caggtaaggt tttttgattt ccactactat gctataataa tgtgtgctgc 5700

aatgctcgtt ttgccatttt aggaaaactg tttctcacat tgaatcatgt cagcatttgt 5760

ttcaggttaa acctattgca gataatgctt gtatgagttt aagactttaa gtgtaatagt 5820

aatttgttat tcttattggc gtataatatg ctgatacctt ttatcccctg cacgcacggg 5880

tttaggtttt ggaactggaa gcaaacaagt cattaaatga agaaaaggat acacctgatc 5940

ggtctccctt gccatggcat atactatttg atcagcagag aaagcaaatt atcatgttat 6000

ggcatttatg ccacatatct cttgtgcacc ggacacagtt ttttcttctg ttaggaggag 6060

acccttctga tcagatatat atggaagttg aacttagaag attgactcgg ttagaacagc 6120

acctggcaga gcttgggaat gctagtcctg cacttctagg tgatgagcct gcaggctctg 6180

tttcagcaag gtatgctcgt gtctttgaaa gacatgtgtc ttttttgaca attcctgcta 6240

gtcctgcact tctaggtgtc tttgataatt ccttaaatca agtgtctttg ataaagctcg 6300

tgtatttgaa aaacaggttt attttagaaa attctaataa attagtgcat ggccttgcac 6360

gcttttattt gtataatgag cagagaatag tatattaatc ttttgctatt gattgatcat 6420

caaatgcagc attagagctc tgaagcaaga aagggaacat cttgctagga aggtgaacac 6480

taaacttaca gcagaggaga gggaactgct ttatgcaaaa tgggaagttc ctccagttgg 6540

aaaacaaagg agactgcaat ttgtaaataa attgtggacc gacccttata acatgcaaca 6600

tgtgcaagaa agtgctgaaa ttgtagcaaa gctcattgat ttcagtgtat ctgatgaaaa 6660

cagcaaggat atgattgaat taaacttttc aagccctttt aataagaaaa catgggcggg 6720

ctggaacttt atatcaaatc ttctaaattt gtaaacacat tgttgtagtt gtacgtcatt 6780

tgtagttgag aagagaagta gagaactatc aaatatatat aatatttcga ttccattaac 6840

tgattattag gtgtacaagt ggcatgtttg caagaaagaa agaa 6884