Mutant of soybean GmTic110 gene and application thereof

文档序号：336571 发布日期：2021-12-03 浏览：7次中文

阅读说明：本技术 大豆GmTic110基因的突变体及其应用 (Mutant of soybean GmTic110 gene and application thereof ) 是由于慧冯献忠杨素欣王秋实张芷睿杨欣晶于 2021-07-06 设计创作，主要内容包括：本发明涉及基因工程技术领域,尤其涉及大豆GmTic110基因的突变体及其应用。本发明研究表明,大豆编码叶绿体膜蛋白的GmTic110基因突变,导致突变体叶绿素合成下降,叶绿体发育异常,影响了植株的光合作用。利用CRISPR/Cas9系统对野生型进行GmTic110基因的敲除,获得稳定的敲除转基因株系表现叶色黄化,与突变体表型一致。本发明为大豆叶绿体工程研究提供基因资源,可通过GmTIC110蛋白定向改造大豆叶绿体发育,提高大豆的光合作用,为高光效大豆品种培育提供种质资源,具有广阔的应用前景。(The invention relates to the technical field of genetic engineering, in particular to a soybean GmTic110 gene mutant and application thereof. The research of the invention shows that the GmTiC110 gene mutation of soybean coding chloroplast membrane protein causes the chlorophyll synthesis of a mutant to be reduced and chloroplast to be abnormal in development, thereby influencing the photosynthesis of plants. The GmTic110 gene is knocked out of a wild type by using a CRISPR/Cas9 system, and a stable knocked-out transgenic strain is obtained to express leaf yellowing and is consistent with a mutant phenotype. The invention provides gene resources for soybean chloroplast engineering research, can directionally modify soybean chloroplast development through the GmTIC110 protein, improves the photosynthesis of soybeans, provides germplasm resources for high-photosynthetic-efficiency soybean variety cultivation, and has wide application prospect.)

1. A soybean GmTic110 gene mutant is characterized in that G at the 341 th site is mutated into A.

2. The mutant according to claim 1, wherein the soybean GmTic110 gene has a sequence shown in SEQ ID No. 1.

3. The mutant according to claim 2, wherein the sequence of the mutant has:

(I) a nucleotide sequence shown as SEQ ID No. 8; or

(II) a complementary nucleotide sequence of the nucleotide sequence shown as SEQ ID No. 8; or

(III) a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or

(IV) nucleotide sequence obtained by replacing, deleting or adding a nucleotide sequence with the nucleotide sequence shown in (I), (II) or (III), and the nucleotide sequence has the same or similar function with the nucleotide sequence shown in (I), (II) or (III); or

(V) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of (I), (II), (III) or (IV).

4. The mutant of claim 3, wherein said substitution, deletion or addition of a nucleotide.

5. A protein encoded by the mutant of any one of claims 1 to 4.

6. The protein of claim 5, having:

(i) and an amino acid sequence shown as SEQ ID No. 9;

(ii) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of (i), and the amino acid sequence has the same function with the amino acid sequence of (i); or

(iii) And (ii) an amino acid sequence having 90% or more identity to the amino acid sequence described in (i) or (ii).

7. The protein of claim 6, wherein said substitution, deletion or addition of an amino acid.

8. Biological material comprising a mutant according to any one of claims 1 to 4 or a protein according to any one of claims 5 to 7;

the biological material comprises one or more of a recombinant expression vector, a plasmid, an expression cassette, a recombinant bacterium or a host cell.

9. Use of a mutant according to any one of claims 1 to 4, a protein according to any one of claims 5 to 7, a biological material according to claim 8, in any one or more of the following:

(A) the application of the compound in the regulation of chlorophyll; and/or

(B) The application in regulating and controlling the development of chloroplasts; and/or

(D) The application in preparing transgenic plants; and/or

(E) And the use thereof for protein subcellular localization.

10. The use of claim 9, wherein the plant is a legume.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a soybean GmTic110 gene mutant and application thereof.

Background

Chlorophyll is one of important pigments involved in photosynthesis in chloroplasts, and research shows that the development condition of plant chloroplasts is in positive correlation with the chlorophyll content in leaves and the photosynthesis rate of the leaves. The mutation of the chlorophyll synthesis related gene can directly or indirectly influence the biosynthesis or degradation pathway of the chlorophyll synthesis related gene, so that the chlorophyll of the plant is deleted, and the plant leaves have chlorophyll deletion type mutant characteristics such as yellowing, whitening, stripe leaf spots, purple brown spots and the like. Chlorophyll-deficient mutants often affect the rate of photosynthesis in plants, causing plant chloroplast to develop slowly, resulting in reduced crop yield and, in severe cases, plant death.

Plant chloroplasts synthesize important amino acid substances through photosynthesis, are main sources of plant cell energy, play an important role in the processes of plant growth and development and cell metabolism, and wherein, inner membrane protein (TIC) plays an important role in substance transportation inside and outside the chloroplasts. Currently, identified chloroplast inner membrane proteins mainly include proteins such as Tic20, Tic21, Tic110 and Tic 236. The Tic110 protein is an important chloroplast inner membrane protein, which is one of the main components of Tic complex and has the property of cation selective channel. Research results show that the TIC110 protein plays an important role in interaction with some large molecules or small molecules of proteins in plants; the TIC110 protein is reported to interact with TIC40 protein to form a complex that transports proteins inside and outside chloroplasts; the interaction of the TIC110 protein and the TIC32 protein plays a redox function and can regulate Ca in chloroplasts²⁺A steady state of ions; the TIC110 protein interacts with a plurality of stroma molecular chaperone Hsp93 and Hsp70 to form a chloroplast intima transport channel bracket, so that various proteins are smoothly guided into chloroplasts to complete biological functions.

Disclosure of Invention

In view of the above, the invention provides a soybean GmTic110 gene mutant and an application thereof. The research of the invention shows that the GmTiC110 gene mutation of soybean coding chloroplast membrane protein causes the chlorophyll synthesis of a mutant to be reduced and chloroplast to be abnormal in development, thereby influencing the photosynthesis of plants. The GmTic110 gene is knocked out of a wild type by using a CRISPR/Cas9 system, and a stable knocked-out transgenic strain is obtained to express leaf yellowing and is consistent with a mutant phenotype. The invention provides gene resources for soybean chloroplast engineering research, can directionally modify soybean chloroplast development through the GmTIC110 protein, improves the photosynthesis of soybeans, provides germplasm resources for high-photosynthetic-efficiency soybean variety cultivation, and has wide application prospect.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a soybean GmTic110 gene mutant, wherein the 341 th G is mutated into A.

In some embodiments of the invention, the sequence of the soybean GmTiC110 gene is shown in SEQ ID No. 1.

In some embodiments of the invention, the sequence of the mutant has:

(I) a nucleotide sequence shown as SEQ ID No. 8; or

(II) a complementary nucleotide sequence of the nucleotide sequence shown as SEQ ID No. 8; or

(IV) a nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences with the nucleotide sequence shown in the (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I), (II) or (III); or

(V) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of (I), (II), (III) or (IV).

In some embodiments of the invention, the substitution, deletion or addition of one nucleotide.

The invention also provides proteins encoded by the mutants.

In some embodiments of the invention, the protein has:

(i) and an amino acid sequence shown as SEQ ID No. 9;

(iii) And (ii) an amino acid sequence having 90% or more identity to the amino acid sequence described in (i) or (ii).

In some embodiments of the invention, the substitution, deletion or addition of an amino acid.

In addition, the present invention provides a biomaterial comprising said mutant or said protein; the biological material comprises one or more of a recombinant expression vector, a plasmid, an expression cassette, a recombinant bacterium or a host cell.

More importantly, the invention also provides the use of the mutant, the protein and the biological material in any one or more of the following items:

(A) the application of the compound in the regulation of chlorophyll; and/or

(B) The application in regulating and controlling the development of chloroplasts; and/or

(D) The application in preparing transgenic plants; and/or

(E) And the use thereof for protein subcellular localization.

In some embodiments of the invention, the plant is a legume.

The research of the invention shows that the GmTiC110 gene mutation of soybean coding chloroplast membrane protein causes the chlorophyll synthesis of a mutant to be reduced and chloroplast to be abnormal in development, thereby influencing the photosynthesis of plants. The GmTic110 gene is knocked out of a wild type by using a CRISPR/Cas9 system, and a stable knocked-out transgenic strain is obtained to express leaf yellowing and is consistent with a mutant phenotype. The invention provides gene resources for soybean chloroplast engineering research, can directionally modify soybean chloroplast development through the GmTIC110 protein, improves the photosynthesis of soybeans, provides germplasm resources for high-photosynthetic-efficiency soybean variety cultivation, and has wide application prospect.

Advantageous effects of the present invention include, but are not limited to:

(1) the GmTic110 gene is separated from soybean and is used as an endogenous gene of the soybean, thus having profound significance for the genetic engineering transformation of the soybean and the research of the soybean molecular regulation mechanism.

(2) The research of the invention shows that the soybean coding chloroplast membrane gene mutation can change the normal transportation of soybean chloroplast, can be used for directionally transforming the chloroplast function, provides gene resources for the development research of the soybean chloroplast and provides germplasm resources for the soybean breeding.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the genomic and transcribed sequence of the soybean GmTiC110 gene, where: the exon sequences are in bold, the intron sequences are in italics,indicates a promoter which is a substance capable of promoting the growth of,represents a terminator;

FIG. 2 shows the difference in leaf color phenotype of wild-type Williams82 and the Gstic 110 mutant;

FIG. 3 shows the difference in chlorophyll content between wild-type Williams82 and Gmatic 110 mutant leaves;

FIG. 4 shows the difference in chloroplast development observed by transmission electron microscopy between wild type Williams82 and GmTic110 mutant leaves;

FIG. 5 shows the difference in photosynthesis indices between wild type Williams82 and GmTiC110 mutant plants; wherein FIG. 5(A) shows the net photosynthetic rate differences between wild type Williams82 and GmTic110 mutant plants; FIG. 5(B) shows the difference in stomatal conductance between wild type Williams82 and GmTic110 mutant plants; FIG. 5(C) shows the difference in transpiration rate between Williams82 and GmTic110 mutant plants; FIG. 5(D) shows the intercellular carbon dioxide concentration differences between Williams82 and GmTic110 mutant plants;

FIG. 6 shows that GmTic110 gene is knocked out by using CRISPR/Cas9 system to obtain a knockout transgenic line with stable phenotype in the background of wild type Williams 82;

FIG. 7 shows protein co-localization of GmTiC110 protein in Arabidopsis protoplasts.

Detailed Description

The invention discloses the application of soybean chloroplast membrane protein gene or protein thereof, and the skilled person can appropriately improve the process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention provides a GmTic110 gene, wherein the GmTic110 gene has any one of the following nucleotide sequences:

I) the nucleotide sequence shown as SEQ ID No. 1;

II) amino acid sequence with same or similar functions and with one or more nucleotides substituted, deleted or added in the nucleotide sequence shown as SEQ ID No. 1;

III) and a vector containing the nucleotide sequence shown in SEQ ID No.1 or a fragment thereof.

In the invention, the amino acid sequence of the GmTic110 protein is provided as one of the following sequences:

I) and an amino acid sequence shown as SEQ ID No. 2;

II) and the amino acid sequence with the same or similar functions in the amino acid sequence shown as SEQ ID No.2 by substituting, deleting or adding one or more amino acids;

in the invention, the nucleotide sequence of the GmTiC110 protein is shown as SEQ ID No. 3.

The invention provides a soybean GmTic110 gene mutant which is characterized in that G at the 341 th site in a nucleic acid sequence shown as SEQ ID No.3 is mutated into A.

The invention provides an application of the soybean GmTic110 gene or a biological material containing the gene in chlorophyll regulation.

The invention provides application of the soybean GmTic110 gene or a biological material containing the gene in regulating and controlling chloroplast development.

The invention provides an application of the soybean GmTic110 gene or a biological material containing the gene in regulation and control of photosynthesis.

The invention provides an application of the soybean GmTic110 gene or a biological material containing the gene in preparation of a transgenic plant.

The invention provides an application of the soybean GmTic110 gene or a biological material containing the gene in regulating and controlling a gene expression mode.

The invention provides an application of the soybean GmTic110 gene or a biological material containing the gene in protein subcellular localization.

The invention also provides a target site designed on the GmTiC110 gene by using the CRISPR/Cas9 system for constructing a gene knockout vector, wherein the skeleton vector of the gene knockout vector is VK005-04-soU6-2-GmUbi 3.

The recombinant host containing the expression vector is agrobacterium, and the gene knockout method provided by the invention adopts an agrobacterium-mediated method to transform the gene knockout vector containing the GmTiC110 gene target site into a plant explant. The explant used as recipient material was hypocotyl cells.

Preferably, the sequences of the specific primers of the gene knockout carrier are shown as SEQ ID No.4 and SEQ ID No. 5.

An upstream primer shown as SEQ ID No. 4: GATTGCGGCGGCTGGATACGGCCT

The downstream primer shown in SEQ ID No. 5: AAACAGGCCGTATCCAGCCGCCGC

In the present invention, the plant is a leguminous plant; the plant used for verifying the function of the GmTic110 in the invention is leguminous plant soybean, in particular soybean Williams 82.

The nucleic acid molecule for coding the GmTIC110 protein comprises genomic DNA, cDNA, recombinant DNA or mRNA, hnRNA for coding the GmTIC110 protein; or a nucleic acid molecule which is reverse complementary to the above DNA, cDNA, recombinant DNA or mRNA.

The nucleic acid molecule can be modified or optimized according to actual needs, so that the gene expression is more efficient; for example, the codon of the GmHY2 gene can be changed to conform to the preference of a recipient plant while the amino acid sequence of the GmHY2 gene is maintained according to the preference of the recipient plant. ② or modifying the gene sequence adjacent to the initiating methionine to allow efficient initiation of translation; for example, the modification is carried out using a sequence known to be effective in plants. Connecting with various plant expression promoters to facilitate the expression of the promoter in plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; (iv) introduction of enhancer sequences such as intron sequences (e.g.from Adhl and bronzel) and viral leader sequences (e.g.from TMV, MCMV and AMV).

In the present invention, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The host may be a fungus, a bacterium, an alga or a cell.

For plants not containing GmTIC110, a gene fragment of GmTIC110 may be introduced into plant cells by a chemical method, a shotgun method, microinjection, electroporation, or the like, or a gene fragment of GmTIC110 may be introduced into plant cells by a method such as homologous recombination, zinc finger nuclease, TALEN, CRISPR, or the like.

The invention has the advantages that:

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 isolation and structural analysis of the GmTic110 Gene in Soybean

(1) Isolation of the GmTiC110 Gene

Total RNA was extracted from a soybean variety Williams82, and first strand cDNA was synthesized using the total RNA as a template and oligo (T)17 as a primer. Using the first strand of cDNA as template, respectively using forward primers

(5'-ATGAACCCTTCCACACTCACC-3', shown as SEQ ID No. 6) and a reverse primer (5'-CTAGAATACAAACTTCTCTTCCT-3', shown as SEQ ID No. 7) for PCR amplification. A cDNA fragment of the GmTic110 gene with the length of 2988bp is obtained, and the fragment is connected with a pEasy-Blunt vector (full-scale gold) and named as Blunt-GmTic 110.

The gene sequence of the obtained GmTic110 gene is shown in SEQ ID No.1, and the total length of CDS of the GmTic110 gene is 2988 bp; the amino acid sequence of the protein coded by the gene is shown in SEQ ID No.2, and has 995 amino acids in total.

(2) Structural analysis of GmTiC110 gene

DNA in young leaves of a soybean variety Williams82 is extracted, and a GmTic110 genome fragment is obtained by amplification by taking the genome DNA as a template, wherein the GmTic110 genome sequence is shown as SEQ ID No.3, the total length is 7280bp, and the GmTic110 genome fragment comprises 15 exons and 14 introns (shown as figure 1).

Example 2 isolation, screening and phenotypic characterization of Soybean GmTiC110 Gene mutants

From the laboratory EMS-mutagenized Williams82 mutant library, a leaf yellowing mutant named Gmatic 110-1(G at position 341 in the nucleic acid sequence of GmTic110 gene is mutated to A, as shown in SEQ ID No. 8) was selected. The Gmatic 110-1 mutant exhibited a leaf yellowing phenotype from seedling stage to maturity stage as compared to wild-type Williams82 (FIG. 2). Meanwhile, the allelic mutant which is screened by the mutant library by us according to the genotype is named as Gmatic 110-2, wherein the GmTic110-2 mutant observes a leaf yellowing phenotype (figure 2).

(1) GmTic110 gene regulates chlorophyll synthesis

Since mutant Gmatic 110 showed a leaf yellowing phenotype throughout the growth period, the chlorophyll and carotenoid contents of Williams82, Gmatic 110-1 and Gmatic 110-2 leaves were measured spectrophotometrically, indicating that the contents of chlorophyll a and chlorophyll b in mutant Gmatic 110-1 and Gmatic 110-2 were significantly lower than in the wild type (FIG. 3). This indicates that the GmTic110 gene mutation influences the synthetic pathway of chlorophyll a and chlorophyll b.

Table 1 figure 3 data

(2) GmTic110 gene regulating soybean chloroplast development

To analyze whether the mutant leaf yellowing can cause structural change of chloroplast, the ultrastructure of wild-type Williams82 and mutant Gmatic 110-1 and GmTic110-2 chloroplasts was observed by transmission electron microscopy. Mutant Gmtic110 chloroplasts had an increased number of starch granules and a reduced accumulation of basal granule thylakoids compared to wild-type Williams82 chloroplasts (fig. 4). Because the thylakoid membrane is provided with the photochromic component and the photosynthesis mainly occurs on the thylakoid membrane, the chlorophyll a and the chlorophyll b in the mutant are both obviously reduced, so that the accumulation and thinning of basal granule thylakoid membranes are caused, and the photosynthetic rate is reduced.

(3) GmTiC110 gene for regulating soybean photosynthesis

As the chlorophyll content of the Gmatic 110 mutant is obviously reduced, in order to determine whether the influence on the photosynthesis of the leaves is caused, the photosynthesis intensity indexes of Williams82, Gmatic 110-1 and Gmatic 110-2 are respectively detected; the results show that the mutants GmTic110-1 and GmTic110-2 have a reduced net photosynthetic rate, a reduced stomatal conductance, a reduced transpiration rate, and an increased intercellular CO2 concentration compared to the wild type, as compared to the wild type of Williams82 (FIG. 5). The results show that the reduction of chlorophyll content of the mutant Gmatic 110 obviously influences the photosynthesis capability of the leaves.

Table 2 figure 5 raw data

Williams82	Pn(umol/m2/s)	Tr(mmol/m2/s)	Cleaf(mmol/m2/s)	CO2int(ppm)
					1	17.1	3.53	184.81	224.7
2	15.54	2.92	189.87	239.5
					3	16.69	3.42	203.55	207.8
4	17.28	3.58	205.99	254.1
					5	17.25	3.99	229.77	256.6
6	14.87	3.29	197.86	245.6
					7	19.02	3.67	195.67	221.3
8	15.41	3.79	214.7	235.6
					9	16.06	3.43	203.52	238
10	17.82	3.94	189.69	226.1
					11	18.42	4.02	221.66	244.2
12	19.61	3.58	189.93	211.4
					13	17.22	3.81	195.78	237
14	17.99	4.2	224.49	249.1
					15	16.03	3.71	189.2	242.8
AV	17.08733333	3.658666667	202.4326667	235.5866667
					SD	1.356557969	0.324606722	14.24829437	14.60493393

Table 3 fig. 5 raw data

Table 4 fig. 5 raw data

Gmtic110-2	Pn(umol/m2/s)	Tr(mmol/m2/s)	Cleaf(mmol/m2/s)	CO2int(ppm)
					1	7.76	2.57	150.18	298.6
2	8.96	2.62	157.83	289.9
					3	10.74	2.37	124.19	290.9
4	7.78	2.94	154.31	274.3
					5	7.87	2.69	154.83	299.8
6	8.95	2.93	185.56	304.5
					7	8.42	2.71	161.73	298.7
8	7.41	2.28	133.61	293.7
					9	7.18	2.17	126.56	291.8
10	8.98	2.48	161.07	292.5
					11	7.79	2.17	142.53	295.9
12	7.97	2.26	145.79	310.5
					13	10.73	2	131.88	311.1
14	7.58	2.11	143.77	293.5
					15	7.76	2.08	146.28	291.9
AV	8.392	2.425333333	148.008	295.84
					SD	1.103204423	0.306358023	15.82743829	8.987991989

Table 5 fig. 5 raw data

Example 3 construction of Soybean GmTiC110 Gene knockout vector

A target site PCR amplification target fragment is designed on a GmTiC110 gene transmembrane domain, upstream and downstream primers of a targeting site are dimerized and connected to a VK005-04-soU6-2-GmUbi3 knockout expression vector. The knockout vector is transformed into a coliform strain Trans5 alpha by a freeze-thaw method, and the resistance in Escherichia coli is kanamycin resistance and the resistance in plants is glufosinate ammonium (Bar) resistance.

Example 4 Agrobacterium-mediated transformation of leguminous plants

In this example, an explant of Williams82 containing a knock-out expression vector of the GmTic110 target site was obtained by agrobacterium-mediated transformation of soybean cotyledonary nodes.

(A) Obtaining of Soybean explants

Selecting mature soybean seeds of Williams82 with smooth surface, no damage, no scab and no crack, and sterilizing by chlorine method for 14 h. Ventilating the sterilized seeds on a super clean bench to completely volatilize chlorine, and germinating in a germination culture medium for 6 h. The hypocotyl of soybean 1/2 was removed, and the soybean was cut longitudinally along the hypocotyl, and the remaining hypocotyl was used as a recipient material for Agrobacterium-mediated transformation.

(B) Transformation of soybean

Adopting secondary agrobacterium infection by an agrobacterium-mediated method, and carrying out dark culture on a co-culture medium at 22 ℃ for 5 d; culturing in SI-I culture medium under strong light for 7 days; cutting off big buds of the explant, and culturing in an SI-II culture medium under strong light for 14 d; cutting out cotyledon and hypocotyl of the explant, and subculturing every 14d in SE medium; shearing off about 3cm of cluster buds, and placing the cluster buds into a rooting culture medium for rooting; and transferring the plant with developed root into soil for planting in the RM rooting culture medium. Through Bar resistance detection, 16 resistant plants are obtained through screening. The pods begin to mature after 5 months of cultivation in a climatic chamber and harvest is complete after 6 months.

When harvested seeds of T1 generation were planted in a climatic chamber, a phenotype of leaf yellowing was observed, consistent with the Gmtic110 mutant phenotype (fig. 6).

Example 5 subcellular localization of Soybean GmTIC110 protein

In order to verify the subcellular localization of the GmTic110 protein, the constructed PUC19-GFP-GmTic110 transient expression vector is used for transforming arabidopsis leaf protoplasts by a polyethylene glycol-mediated method, and the coexlocalization observation is carried out by using a Nikon laser confocal microscope, wherein GFP fluorescence is localized on the chloroplast inner membrane (figure 7).

Comparative example

Control group: deletion of one base in the exon of the Gmatic 110 mutant leads to mutation at the 113 th amino acid and premature termination at the 120 th amino acid, and the mutation occurs in the second transmembrane domain, thus leading to yellowing phenotype of leaves;

experimental groups: in the invention, the 1 st exon of the gene in the Gmatic 110-1 mutant is subjected to one base substitution to cause amino acid substitution, the mutation site is generated in the second transmembrane domain, and the 14 th exon of the gene in the Gmatic 110-2 mutant is subjected to one base substitution to cause amino acid substitution, and the site is generated in the binding domain of an interaction factor.

The yellowing phenotype of the leaves of the mutant material is more remarkable than that of the mutant material of a control group, and the comparison between the chlorophyll content and the photosynthesis index is obviously lower than that of the control group. The targeting site of the Gmatic 110-CR mutant obtained by the CRISPR/Cas9 technology obtains mutants with base deletion and substitution in the first transmembrane domain respectively. The phenotype of the transgenic knockout mutant material is also more prominent than the control group phenotype.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> institute of geography and agroecology of northeast China academy of sciences

<120> soybean GmTic110 gene mutant and application thereof

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gttgcaaatt tgcaatagca tcatgggttt tagatgaata atggatactt ggagcttgac 1320

cattgttaga gtctccagtg caggcatttc aaaagttgat atatgtgtca aatcttgtat 1380

ttggagatgc atcgtctttc cttctacctt ggaagcgtgt attcaaggtc accgattccc 1440

aggtatttat ttcttattgg ttaactggta tgcctagctt gtgcaacgtg atattaatat 1500

gcaattagga tgagagattt ggagcatagg tttatgacta tttataggca tttgctcaaa 1560

aagggaagac ctaatttgct gcacaaaatg cattttgcca tgtttgtgat cagtgacatg 1620

ctggcagcat ttcttaataa ttcttatatg ttctcattgt catttcttat cttagtgttt 1680

ttgttatttt tcatttgact agttaactat aatctattta ccagattgaa gtagctgtac 1740

gtgacaatgc gcagcgattg tttgcttcca agctgaaatc agttggcaga ggtaattgtg 1800

tagtcagtaa ttcctcttgg tccttgtttg ctcttactgt aacaaagttg atcaactaac 1860

tcacatttac tcctcgagaa gttgtcataa cttcatacta ctatattact ggagaaatga 1920

ttggaaatga taatcttgtt ttgtaattgt gcatgcattt cttattaaat aaagccttta 1980

aattttgctt gtactcatct agtatgctgg attttgttct gtaattctct agaattacta 2040

tcatccaaac taacaaattc tttctatagc aatttttaga agttttaaat caggtttatt 2100

tcctaagtgt taaccaatga tttcttgcca gatattgatg cagaaaaact tgttgcacta 2160

agaaaagaac aacaattatg tcgcctttct gatgaggttc attttcatta tttcttcttt 2220

cataactttc aacttctctg ggttaaatcc ctgtctttgt cttgccatca aatagttcct 2280

tggtatagta taatcttttt attaattaaa ggaaattttc attgaagctt gctgagaact 2340

tgtttagaga tcacacaagg aaattggttg aggaaaatat ttcagaggca aatagaatac 2400

ttaaatctcg caccaaagca gtgtaagatt catggttttt ctaagtattc ttttttttaa 2460

gtattaaatt ttgaattctg ggcatgtttt tcttgccatt tgtttcatta gtgttactat 2520

tcatttctta atttttacaa aaatagaatt tgccatatct gatcatctgt aagtttcagt 2580

cctggagcta cgcaggcaat tgcagagctg gataaggtat tggcatttaa taatttactc 2640

atctcattta agaatcatcc agatgtggat cgctttgctc gtggtgttgg tccaatttct 2700

ttagtgggta agtgtctgtg actaggtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgta 2760

taacatgttc ttttctcttt ttaatcttaa ttcttgaagt ttaccacaat tcatttttgg 2820

cataggtgga gagtatgatg gtgacagaaa aatagaggac ttgaaactcc tttacagggc 2880

atatgtttca gatgctttgt ctggtgggcg catggaagat gataaggtaa ataaccaaga 2940

caattggata tgaactgtga acttcttaag atgtgttttc tcaatattct aatatatttt 3000

gaagttgagt ttgggttagt gttttccaga tgtatgtagt gattgaatag tatattttct 3060

aaaaaaattg gagtattcag cagctattta tatcatattg gagtattcac ttcccatggg 3120

tataatagag attgaaacct agcagtttag ccttagttat tctttattgt ttcttatgtt 3180

ttatctttct ttttgatgta gtaccaattg ccaaatcatg attgaacagg ctgttcagtg 3240

catgctacca tatccatatt gctcttattc ttatttttaa tatttcagtt agggcagtat 3300

attattttca gtacaagggt atttgtttac tgtatcaagc ctaatgaatt ttcacttatt 3360

gaagcatttt ttagtgttat aaaacaatgt cctgtctcct tgcttaacac taacaactta 3420

aagcttttag gtcaggtgat ttgttgactg ttaagatgta ctagcatacg attccccttt 3480

gaatatagtt gcatatgttc tctaatcttc ctttaatgag tcaacattca gataagcaaa 3540

gacacaacag tgtctcgaaa accatgatat ccattactaa agagttctta ttgatataag 3600

atatgaaact tgatctgtca aaacacttat tttttaatat aaaataaaaa atatatttta 3660

aatatattta ttattcgttg cagcttgctg cactaaatca gttacgaaat atattcggac 3720

tgggtaaacg tgaagccgag gccatttcac ttgatgttac gtcgaaggta tatcgcaaac 3780

gacttgcaca ggctgctgca gatggtgagt tagaaatggc tgatagcaaa gcagccttcc 3840

ttcaaaatct atgtgatgaa cttcattttg atccacaaaa ggccagtgaa cttcatgaag 3900

gtaaaactcg tcatgtttac agttagttgg aagttgacta tatttgaggc ctttattatt 3960

gaacacttta tgtgctgtcg gtttttattt atgtgcaatt cttaaatctt aaggttgtaa 4020

aatgtaaact tgtaaaaccc tttctacatg atgtgacttt tttttatata tatatttagt 4080

tataacacaa tctaatgata gtttattatt tttaagaaat ttatcggcaa aagcttcaga 4140

gatgtgtagc tgatggggag ctcaatgagg aggatgttgc tgctttgttg aggatgcgtg 4200

taatgctctg cataccgcaa cagattgttg aagcagctca ttcagatatc tgtggcagtt 4260

tgtttgaaaa ggtagtcaac gttacttttt cctatctaat tgtttggtcc ttcataagtg 4320

tcaaattagt gaaattacat atttcttgct atgcagaagt gcggtccatc catgcactgt 4380

aacaaactga taatgataat atgaatacat ggatgaacac taaaattgct ttacatatga 4440

atatgggttc aagatgatta tggaactatg gttatcaaac tcattctact ttctatttga 4500

atattggtag catgagaagt tacagctgtc acattttaat gtgttgataa acctgataat 4560

gctttcattt actggtagtg tggagatgga gaccatgaat ctactgcatt tttttttatc 4620

ttacaggaaa gatacactgt tgtactctgt actctgcatg ctaggttttg tctagaattt 4680

ctaaaatgtg tatctattgg aattgttact ttcaactgct tgtaatttat ttgggctttg 4740

tcttttcaat ttgtagacct gtatctactt gctataaaat ctgtttattg gtttgattat 4800

cttatatcaa agttgtacaa gtctagattc tatagtgctt agttgatggc agcgatatgt 4860

gaagaaatgg gtctgtttta ttttgatata tcatgagtct tgtttcaatc tccatcaaag 4920

atgttcaaaa ttttgttgag ttattactaa ttcatgtttc tccttttata ggttgtcaag 4980

gaggcaattg catcaggggt tgatggatat gatgctgaaa tccagaaatc agtaagaaaa 5040

gcagcacatg gcctgcgact tactagggag gttgctatgt ctattgcaag caaggcggtg 5100

agtatcaagt tatcagaaac tcaatggtga tttataaaga tttgaatagg tttaaatttc 5160

actgaggttt tttggaatta taggtaagga agatttttat taattacata aaacgtgcac 5220

gggcagctgg aaatcgtacc gagtctgcga aagaactgaa gaagatgata gccttcaaca 5280

ccttagttgt aactaatttg gtggaggaca ttaaagggga gtcaactgat atttcctctg 5340

aagaacctgt gaaagaggac attacacaaa ctgacgatga agaatgggaa tcacttcaga 5400

cactcaagaa aataagacca aataaagaac ttacggaaaa gttgggaaaa cctggtcaga 5460

cagaaattac tctgaaagat gatcttcctg aaagggatag gactgatctt tacaagacat 5520

acttacttta ttgtctaact ggtgaagtga caagggttcc atttggtgct cagatcacta 5580

ctaagaagga tgattctgag tatcttcttc taaatcagct tggtgggatc ctgggattga 5640

gtagtcaaga aatagtggaa gtgcacaggg gtctagctga gcaggctttt aggcaacagg 5700

ctgaggtaat tttagccgat ggacagttga caaaggccag ggtggagcag cttaataacc 5760

tccagaaaca agtaggctta cctcaagaat atgctcagaa aataatcaag agtataacca 5820

ctacaaaaat ggcagctgcc attgaaactg ctgtaactca agggaggctc aatatgaagc 5880

agataaggga acttaaggaa gctgatgttg atttagacag tatggtatct gagaacttga 5940

gagagaccct cttcaaaaaa actgttgatg acattttctc atccggtact ggagagtttg 6000

acactgagga agtatatgaa aaaatcccgt cagatctcaa cattaacaaa gagaaggcac 6060

gaggtgttgt tcatgagctt gcaaagggta gactatccaa ctctctgatt caggctgtct 6120

ctctactaag acagagaaat cagcagggag tggtaagtaa tattcaaagc ttcatgttca 6180

cttagtacta gtttggattt atgctggatt atctaaaatt acactcaaat actagatcac 6240

gtgaagtaaa tgttcataaa ttttttgttt cactttaaga aattgattct gagttgaagc 6300

aattttaggt aactttttct gttgaataaa aaaggtttat gttaagtttt attattaact 6360

tgctttcatt aaaaaaaaac ctccaaacat aaatcttttc acttcaaaat caattatgca 6420

aaatctactt cattcataat caattttgca aatgtttgtc ccaacacact tagtgcgtgt 6480

ttgtacaagc attggcaaat tgattttaaa ttgaattgat tttagttaaa ttgatttgaa 6540

agtgatgtga tttatgttta agtacaacat tttagatcca tcgtaaaaat tactcaaagg 6600

tcctttaact tagagtcaat tttggactta gaatcaattc tgaattcttc tttaacagga 6660

aaccaaacac gaaaatttat caaaaagcca ttctccaatg tgaaacaaaa cttgcactta 6720

ttcaaacagc ttttttacgt ttgtttgtca tggataatca tgtgtgattg cttcttttct 6780

tttgtgcagg tttcttcact caatgacttg ctggcatgtg acaaagcagt accctcacag 6840

ccagtttcat gggaagtgcc agaggagctt tctgatctat acaccatata cttgaagagt 6900

aatccaactc ctgagaattt gtctcgtttg caatatctgt tgggtataaa tgattccaca 6960

gctgctgctc ttagggagat tggagataga ttactcaata ctactgcgga ggaagagaag 7020

tttgtattct agtcttgtga taaaatgttt ggcattttag gcacactaga gaggctgcaa 7080

gtccccttaa ttttgccatt ttttattttc cattttgttt aacggtagca gaaaaagaat 7140

atcacgatgt ataaactttg gaatagagat gagtgcagtg ctgaaatctt agataatatt 7200

ttgtttgaga cagttgaatt aatcaattgc tattttttat gaatgaaaat aagttttcat 7260

aatcgtggac aagttttatc 7280

<210> 2

<211> 995

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Asn Pro Ser Thr Leu Thr Pro Ser His Thr His Arg Pro Leu Leu

1 5 10 15

Leu Pro Ser Pro Phe His Thr Arg Arg Arg Arg Phe Lys Val Ser Leu

20 25 30

Pro Arg Cys Ser Ser Ser Ser Ala Ala Ser Ser Pro Pro Pro Pro Pro

35 40 45

Pro Pro Pro Pro Gln Arg Pro Pro Lys Asp Leu Lys Gly Ile Asp Val

50 55 60

Leu Val Asp Lys Leu Ser Pro Pro Ala Arg Leu Ala Thr Ser Ala Val

65 70 75 80

Ile Val Ala Gly Ala Ala Ala Ala Gly Tyr Gly Leu Gly Ser Arg Phe

85 90 95

Gly Gly Ser Arg Tyr Ala Ala Leu Gly Gly Ala Val Ala Leu Gly Ala

100 105 110

Ala Gly Gly Ala Ala Ala Tyr Ala Leu Asn Ala Ala Ala Pro Gln Val

115 120 125

Ala Ala Val Asn Leu His Asn Tyr Val Ala Ala Phe Asp Asp Pro Ser

130 135 140

Lys Leu Lys Lys Glu Glu Ile Glu Ala Ile Ala Ser Lys Tyr Gly Val

145 150 155 160

Ser Lys Gln Asp Glu Ala Phe Lys Thr Glu Ile Cys His Ile Tyr Ser

165 170 175

Glu Phe Val Ser Ser Val Leu Pro Pro Gly Gly Glu Glu Leu Lys Gly

180 185 190

Asp Glu Val Asp Arg Ile Val Ser Phe Lys Asn Ser Leu Gly Ile Asp

195 200 205

Asp Pro Asp Ala Ala Ala Met His Met Glu Ile Gly Arg Lys Phe Phe

210 215 220

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

225 230 235 240

Arg Ala Phe Gln Lys Leu Ile Tyr Val Ser Asn Leu Val Phe Gly Asp

245 250 255

Ala Ser Ser Phe Leu Leu Pro Trp Lys Arg Val Phe Lys Val Thr Asp

260 265 270

Ser Gln Ile Glu Val Ala Val Arg Asp Asn Ala Gln Arg Leu Phe Ala

275 280 285

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

290 295 300

Ala Leu Arg Lys Glu Gln Gln Leu Cys Arg Leu Ser Asp Glu Leu Ala

305 310 315 320

Glu Asn Leu Phe Arg Asp His Thr Arg Lys Leu Val Glu Glu Asn Ile

325 330 335

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

340 345 350

Ala Thr Gln Ala Ile Ala Glu Leu Asp Lys Val Leu Ala Phe Asn Asn

355 360 365

Leu Leu Ile Ser Phe Lys Asn His Pro Asp Val Asp Arg Phe Ala Arg

370 375 380

Gly Val Gly Pro Ile Ser Leu Val Gly Gly Glu Tyr Asp Gly Asp Arg

385 390 395 400

Lys Ile Glu Asp Leu Lys Leu Leu Tyr Arg Ala Tyr Val Ser Asp Ala

405 410 415

Leu Ser Gly Gly Arg Met Glu Asp Asp Lys Leu Ala Ala Leu Asn Gln

420 425 430

Leu Arg Asn Ile Phe Gly Leu Gly Lys Arg Glu Ala Glu Ala Ile Ser

435 440 445

Leu Asp Val Thr Ser Lys Val Tyr Arg Lys Arg Leu Ala Gln Ala Ala

450 455 460

Ala Asp Gly Glu Leu Glu Met Ala Asp Ser Lys Ala Ala Phe Leu Gln

465 470 475 480

Asn Leu Cys Asp Glu Leu His Phe Asp Pro Gln Lys Ala Ser Glu Leu

485 490 495

His Glu Glu Ile Tyr Arg Gln Lys Leu Gln Arg Cys Val Ala Asp Gly

500 505 510

Glu Leu Asn Glu Glu Asp Val Ala Ala Leu Leu Arg Met Arg Val Met

515 520 525

Leu Cys Ile Pro Gln Gln Ile Val Glu Ala Ala His Ser Asp Ile Cys

530 535 540

Gly Ser Leu Phe Glu Lys Val Val Lys Glu Ala Ile Ala Ser Gly Val

545 550 555 560

Asp Gly Tyr Asp Ala Glu Ile Gln Lys Ser Val Arg Lys Ala Ala His

565 570 575

Gly Leu Arg Leu Thr Arg Glu Val Ala Met Ser Ile Ala Ser Lys Ala

580 585 590

Val Arg Lys Ile Phe Ile Asn Tyr Ile Lys Arg Ala Arg Ala Ala Gly

595 600 605

Asn Arg Thr Glu Ser Ala Lys Glu Leu Lys Lys Met Ile Ala Phe Asn

610 615 620

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

625 630 635 640

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

645 650 655

Asp Glu Glu Trp Glu Ser Leu Gln Thr Leu Lys Lys Ile Arg Pro Asn

660 665 670

Lys Glu Leu Thr Glu Lys Leu Gly Lys Pro Gly Gln Thr Glu Ile Thr

675 680 685

Leu Lys Asp Asp Leu Pro Glu Arg Asp Arg Thr Asp Leu Tyr Lys Thr

690 695 700

Tyr Leu Leu Tyr Cys Leu Thr Gly Glu Val Thr Arg Val Pro Phe Gly

705 710 715 720

Ala Gln Ile Thr Thr Lys Lys Asp Asp Ser Glu Tyr Leu Leu Leu Asn

725 730 735

Gln Leu Gly Gly Ile Leu Gly Leu Ser Ser Gln Glu Ile Val Glu Val

740 745 750

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

755 760 765

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

770 775 780

Leu Gln Lys Gln Val Gly Leu Pro Gln Glu Tyr Ala Gln Lys Ile Ile

785 790 795 800

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

805 810 815

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

820 825 830

Asp Val Asp Leu Asp Ser Met Val Ser Glu Asn Leu Arg Glu Thr Leu

835 840 845

Phe Lys Lys Thr Val Asp Asp Ile Phe Ser Ser Gly Thr Gly Glu Phe

850 855 860

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

865 870 875 880

Lys Glu Lys Ala Arg Gly Val Val His Glu Leu Ala Lys Gly Arg Leu

885 890 895

Ser Asn Ser Leu Ile Gln Ala Val Ser Leu Leu Arg Gln Arg Asn Gln

900 905 910

Gln Gly Val Val Ser Ser Leu Asn Asp Leu Leu Ala Cys Asp Lys Ala

915 920 925

Val Pro Ser Gln Pro Val Ser Trp Glu Val Pro Glu Glu Leu Ser Asp

930 935 940

Leu Tyr Thr Ile Tyr Leu Lys Ser Asn Pro Thr Pro Glu Asn Leu Ser

945 950 955 960

Arg Leu Gln Tyr Leu Leu Gly Ile Asn Asp Ser Thr Ala Ala Ala Leu

965 970 975

Arg Glu Ile Gly Asp Arg Leu Leu Asn Thr Thr Ala Glu Glu Glu Lys

980 985 990

Phe Val Phe

995

<210> 3

<211> 2988

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgaaccctt ccacactcac cccttcccac acccaccgtc ctctcctcct accctctccc 60

ttccacacca gacggcgtcg ttttaaagtc tccctccctc gctgttcctc ctcctccgcc 120

gcctcctccc cccctcctcc gccacctccg ccgccgcagc gaccgcccaa ggacctcaag 180

ggaatcgatg tcctcgtcga caagctctcg ccgccggcca ggctcgccac ctccgccgtc 240

atcgtcgccg gcgccgcggc ggctggatac ggcctcggct cccgcttcgg cggaagccgc 300

tacgctgcgc tcggtggagc tgtcgccctc ggcgcggccg gcggtgctgc ggcgtacgct 360

ctgaacgcgg ctgctccgca agtcgccgcc gtgaatttgc acaactacgt cgccgcgttc 420

gacgaccctt cgaagctgaa gaaggaagag attgaggcca ttgcgtccaa gtatggtgtg 480

agcaagcaag atgaggcatt caaaactgag atttgtcata tatattccga gtttgtatct 540

tctgtgcttc ctcctggtgg tgaggaactt aaaggtgatg aggttgatag gattgtcagc 600

ttcaaaaatt ctttgggaat tgatgacccg gatgctgctg ctatgcacat ggagattggg 660

aggaaatttt tcaggcaaag gttggaagtt ggggatcgtg atgctgatgt tgagcaacgt 720

cgggcatttc aaaagttgat atatgtgtca aatcttgtat ttggagatgc atcgtctttc 780

cttctacctt ggaagcgtgt attcaaggtc accgattccc agattgaagt agctgtacgt 840

gacaatgcgc agcgattgtt tgcttccaag ctgaaatcag ttggcagaga tattgatgca 900

gaaaaacttg ttgcactaag aaaagaacaa caattatgtc gcctttctga tgagcttgct 960

gagaacttgt ttagagatca cacaaggaaa ttggttgagg aaaatatttc agaggcaaat 1020

agaatactta aatctcgcac caaagcagtt cctggagcta cgcaggcaat tgcagagctg 1080

gataaggtat tggcatttaa taatttactc atctcattta agaatcatcc agatgtggat 1140

cgctttgctc gtggtgttgg tccaatttct ttagtgggtg gagagtatga tggtgacaga 1200

aaaatagagg acttgaaact cctttacagg gcatatgttt cagatgcttt gtctggtggg 1260

cgcatggaag atgataagct tgctgcacta aatcagttac gaaatatatt cggactgggt 1320

aaacgtgaag ccgaggccat ttcacttgat gttacgtcga aggtatatcg caaacgactt 1380

gcacaggctg ctgcagatgg tgagttagaa atggctgata gcaaagcagc cttccttcaa 1440

aatctatgtg atgaacttca ttttgatcca caaaaggcca gtgaacttca tgaagaaatt 1500

tatcggcaaa agcttcagag atgtgtagct gatggggagc tcaatgagga ggatgttgct 1560

gctttgttga ggatgcgtgt aatgctctgc ataccgcaac agattgttga agcagctcat 1620

tcagatatct gtggcagttt gtttgaaaag gttgtcaagg aggcaattgc atcaggggtt 1680

gatggatatg atgctgaaat ccagaaatca gtaagaaaag cagcacatgg cctgcgactt 1740

actagggagg ttgctatgtc tattgcaagc aaggcggtaa ggaagatttt tattaattac 1800

ataaaacgtg cacgggcagc tggaaatcgt accgagtctg cgaaagaact gaagaagatg 1860

atagccttca acaccttagt tgtaactaat ttggtggagg acattaaagg ggagtcaact 1920

gatatttcct ctgaagaacc tgtgaaagag gacattacac aaactgacga tgaagaatgg 1980

gaatcacttc agacactcaa gaaaataaga ccaaataaag aacttacgga aaagttggga 2040

aaacctggtc agacagaaat tactctgaaa gatgatcttc ctgaaaggga taggactgat 2100

ctttacaaga catacttact ttattgtcta actggtgaag tgacaagggt tccatttggt 2160

gctcagatca ctactaagaa ggatgattct gagtatcttc ttctaaatca gcttggtggg 2220

atcctgggat tgagtagtca agaaatagtg gaagtgcaca ggggtctagc tgagcaggct 2280

tttaggcaac aggctgaggt aattttagcc gatggacagt tgacaaaggc cagggtggag 2340

cagcttaata acctccagaa acaagtaggc ttacctcaag aatatgctca gaaaataatc 2400

aagagtataa ccactacaaa aatggcagct gccattgaaa ctgctgtaac tcaagggagg 2460

ctcaatatga agcagataag ggaacttaag gaagctgatg ttgatttaga cagtatggta 2520

tctgagaact tgagagagac cctcttcaaa aaaactgttg atgacatttt ctcatccggt 2580

actggagagt ttgacactga ggaagtatat gaaaaaatcc cgtcagatct caacattaac 2640

aaagagaagg cacgaggtgt tgttcatgag cttgcaaagg gtagactatc caactctctg 2700

attcaggctg tctctctact aagacagaga aatcagcagg gagtggtttc ttcactcaat 2760

gacttgctgg catgtgacaa agcagtaccc tcacagccag tttcatggga agtgccagag 2820

gagctttctg atctatacac catatacttg aagagtaatc caactcctga gaatttgtct 2880

cgtttgcaat atctgttggg tataaatgat tccacagctg ctgctcttag ggagattgga 2940

gatagattac tcaatactac tgcggaggaa gagaagtttg tattctag 2988

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gattgcggcg gctggatacg gcct 24

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aaacaggccg tatccagccg ccgc 24

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgaaccctt ccacactcac c 21

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctagaataca aacttctctt cct 23

<210> 8

<211> 7280

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

acgacgttat ctacaatcta caaagaggct ctatcatctt cttcctcttt agttttttcg 60

ttaaacccta taataacaac cactccactc ttctctactc tccgcaacct tacgacgtcg 120

tctcaaccat gaacccttcc acactcaccc cttcccacac ccaccgtcct ctcctcctac 180

cctctccctt ccacaccaga cggcgtcgtt ttaaagtctc cctccctcgc tgttcctcct 240

cctccgccgc ctcctccccc cctcctccgc cacctccgcc gccgcagcga ccgcccaagg 300

acctcaaggg aatcgatgtc ctcgtcgaca agctctcgcc gccggccagg ctcgccacct 360

ccgccgtcat cgtcgccggc gccgcggcgg ctggatacgg cctcggctcc cgcttcggcg 420

gaagccgcta cgctgcgctc ggtggagctg tcgccctcgg cgcggccgac ggtgctgcgg 480