CN109161551B - Cabbage BoMS1 gene and application thereof in creating sterile materials - Google Patents

Abstract

The invention belongs to the field of biotechnology breeding, and particularly relates to the cabbage BoMS1 gene and its application in creating sterile materials. It provides the cabbage BoMS1 gene, a key gene for controlling male sterility in cabbage, and down-regulates the expression of the cabbage BoMS1 gene through RNAi technology, thereby creating a transgenic male sterile plant line, which overcomes the use of male sterility. When breeding, it relies heavily on a single ogura CMS single sterile resource, and there is a problem of genetic vulnerability risk; it uses RNAi technology to down-regulate the expression of the cabbage BoMS1 gene and the cabbage SRK gene at the same time, creating a hybrid compatibility with the maintainer line during flowering. The transgenic male sterile line created by this method avoids the problem of artificial bud stage pollination for the reproduction of the maintainer line, and reduces the reproductive cost of the male sterile line of cabbage; it also mutates the BoMS1 gene and Cabbage SRK gene, created a non-transgenic male sterile line compatible with the maintainer line at flowering.

Description

Cabbage BoMS1 Gene and Its Application in Creating Sterile Materials

technical field

The invention belongs to the field of biotechnology breeding, and particularly relates to the cabbage BoMS1 gene and its application in creating sterile materials.

Background technique

I.et al.Environmental factors and genotypicvariation of self-incompatibility in Brassica oleracea L.var.Capitata.ActaBiologica Cracoviensia-Series Botanica，2003，45：49-52；Liu LW et al.Evaluationof genetic purity of F1hybrid seeds in cabbage with RAPD,ISSR,SRAP,and SSRmarkers.2007，HORTSCIENCE 42(3):724–727)。Cabbage is one of the cruciferous vegetables widely grown in the world and one of the important autumn and winter leafy vegetables in my country. The annual planting area of cabbage in China is about 12 million mu. The first-generation hybrid varieties can greatly improve the yield and stress resistance of cabbage. The promotion and utilization of the first-generation hybrid varieties is an important way to improve the production efficiency of cabbage. For a long time, the breeding of first-generation cabbage hybrids was mainly achieved by using the self-incompatibility approach, that is, using two different S-haplotype self-incompatibility lines at flowering through bee pollination. The seeds received on one self-incompatible line were hybrid generation seeds, and the propagation of the two self-incompatible parents was accomplished by artificial self-pollination at the bud stage. Since self-incompatibility is easily disturbed by environmental factors such as high temperature and drought, the phenomenon of incompatibility during flowering occurs, resulting in purity problems in hybrid seeds, economic losses to cabbage producers, and the phenomenon of parent loss (

I.et al.Environmental factors and genotypicvariation of self-incompatibility in Brassica oleracea L.var.Capitata.ActaBiologica Cracoviensia-Series Botanica, 2003, 45:49-52; Liu LW et al.Evaluationof genetic purity of F1hybrid seeds in cabbage with RAPD, ISSR, SRAP, and SSRmarkers. 2007, HORTSCIENCE 42(3):724–727).

Breeding the first-generation hybrid varieties of cabbage with male-sterile lines can solve the above problems. male sterility in vegetables for hybrid development. AReview. Adv. Hort. Sci., 2010, 24(4):263-279). Currently, the breeding of first-generation cabbage vegetable hybrids based on the male-sterile pathway relies heavily on the male-sterile male-sterile lines of ogura CMS from radish (Parkash C etal.'Ogura'-based'CMS'lines with different nuclear Backgrounds of cabbagerevealed substantial diversity at morphological and molecularlevels. 3 Biotech. 2018, 8: 27; Shu JS et al. Detection of the diversity of cytoplasmic male sterility sources in Broccoli (Brassica Oleracea var. Italica) using mitochondrial markers. Front Plant Sci. 2016; 7:927.), and the widespread use of single cytoplasm in variety breeding will lead to genetic vulnerability of varieties and bring potential ecological risks (Tatum LA. The southern corn leaf blight epidemic. Science, 1971, 171 (3976) :1113-1116). Genetic mutations during plant pollen development often lead to complete male sterility, resulting in plant male sterile lines controlled by recessive nuclear genes (Chang F et al. Molecular control of microsporogenesis in Arabidopsis. Current Opinion in Plant Biology 2011, 14: 66–73 ). Therefore, site-directed mutation of genes involved in the development of cabbage pollen will be able to produce male sterility, which is beneficial to expand male sterility resources in cabbage, and is important to solve the risk of using a single ogura CMS male sterility source in current cabbage breeding. the value of.

Although the breeding of the first generation of cabbage hybrids through the male sterility approach can ensure the purity of hybrid seeds, due to the influence of the self-incompatibility of cabbage, the sterile line, maintainer line and the reproduction of the male parent line in this breeding approach still need to rely on In the traditional artificial pollination at the bud stage, the propagation cost of the entire hybrid seed is even higher than the cost of producing hybrid seeds using self-incompatible lines. Therefore, while creating a sterile line of cabbage, the gene that controls the self-incompatibility SRK in the sterile line is down-regulated or mutated, and the reproductive cost of the male sterile line of cabbage is reduced by pollination and hybridization of vector insects such as bees during flowering. The whole seed production cost of the first-generation seeds of cabbage hybrids selected based on the male sterility approach is reduced.

SUMMARY OF THE INVENTION

In order to solve the problem of genetic vulnerability risk due to a large amount of dependence on a single oguraCMS single sterile resource when the male sterility approach is used to carry out the breeding of the first generation of cabbage hybrids, the present invention provides a key gene BoMS1 for controlling male sterility in cabbage, Corresponding transgenic male sterile lines and non-transgenic male sterile lines can be obtained by down-regulating the expression of BoMS1 or mutating the BoMS1 gene.

Cabbage BoMS1 gene, the amino acid sequence of the encoded protein is shown in SEQ ID No.1.

Preferably, the cabbage BoMS1 gene contains the nucleotide sequence shown in SEQ ID No.2.

Preferably, the nucleotide sequence of the cabbage BoMS1 gene is shown in SEQ ID No.3.

The application of down-regulating the expression of the above-mentioned cabbage BoMS1 gene or mutating the above-mentioned cabbage BoMS1 gene in the creation of a male sterile plant line.

Down-regulating the expression of the above-mentioned cabbage BoMS1 gene mainly includes the following methods (1) RNA interference, wherein, the production of the RNAi hairpin structure is not limited to the use of the first exon and the first intron of the BoMS1 gene, but also includes any use of the gene. RNAi made from a sequence (>25nt in length); (2) antisense RNA technology; (3) other pathways that can lead to the decline of the BoMS1 gene.

The method for mutating the above-mentioned Brassica oleracea BoMS1 gene includes the use of CRISPR/Cas9, TALE, ZFN and other gene editing technologies, chemical mutagenesis and physical mutagenesis, etc. to achieve the Brassica oleracea BoMS1 gene mutation.

The present invention also provides an iBoMS1 that downregulates the expression of the above-mentioned Brassica oleracea BoMS1 gene, the nucleotide sequence of which is shown in SEQ ID No.4.

Gene expression cassette containing iBoMS1 described above.

Preferably, in the gene expression cassette containing iBoMS1, a promoter PBoMS1 is inserted upstream of iBoMS1, and the nucleotide sequence of PBoMS1 is shown in SEQ ID No.5.

The above-mentioned plant expression vector containing the gene expression cassette of iBoMS1 is inserted.

Preferably, the above-mentioned plant expression vector is further inserted with an iSRK3 gene expression cassette, the iSRK3 gene expression cassette contains iSRK3, and the nucleotide sequence of the iSRK3 is shown in SEQ ID No.8.

More preferably, the SLG13 promoter is inserted upstream of the iSRK3, and the nucleotide sequence of the SLG13 promoter is shown in SEQ ID No.9.

Using the above-mentioned plant expression vector to create a transgenic male sterile line, the plant expression vector inserted with the iBoMS1 gene expression cassette was transformed into cabbage by Agrobacterium-transfected cabbage hypocotyl transformation method to obtain a transgenic male sterile line.

In order to solve the characteristics of self-incompatibility in the breeding of male sterile lines of cabbage, the breeding of sterile line seeds requires artificial bud pollination due to hybrid incompatibility with the corresponding maintainer line at the flowering stage, resulting in seed production costs. To add to the problem, the present invention also provides a method for creating a male sterile line that is cross-compatible with the maintainer line during flowering.

The method is achieved by simultaneously down-regulating the expression of the cabbage BoMS1 gene and the cabbage SRK gene or simultaneously mutating the cabbage BoMS1 gene and the cabbage SRK gene.

The above-mentioned cabbage SRK gene refers to the cabbage S-site receptor kinase gene.

The above-mentioned down-regulation of the expression of the cabbage BoMS1 gene and the cabbage SRK gene at the same time mainly includes the following methods: (1) RNA interference, wherein, the making of the RNAi hairpin structure is not limited to using the first exon and the first intron of the BoMS1 gene, but also Including RNAi made by using any sequence of the gene (length>25nt); (2) using antisense RNA technology; (3) other methods that can lead to the decrease of BoMS1 gene.

The above-mentioned method for simultaneously mutating the cabbage BoMS1 gene and the cabbage SRK gene includes the use of CRISPR/Cas9, TALEN, ZFN and other gene editing technologies, chemical mutagenesis and physical mutagenesis, etc. to achieve the mutation of the cabbage BoMS1 gene and the SRK gene.

The method for creating a male sterile line that is hybrid compatible with the maintainer line at flowering stage is as follows: the plant expression vector inserted with the iBoMS1 gene expression cassette and the iSRK3 gene expression cassette is transfected into cabbage by Agrobacterium-transfected cabbage hypocotyl transformation method, A transgenic male sterile line that is cross-compatible with the maintainer line during flowering is obtained.

The method for creating a male sterile line that is hybrid compatible with the maintainer line at flowering stage is as follows: according to the requirements of the sequence characteristics of the target site of the CRISPR/Cas9 gene editing system, the exon of the cabbage BoMS1 gene and the exon of the cabbage SRK3 gene are respectively selected. Select 4 target sites with a length of 20bp (less than 4 target sites can be selected in actual operation), the downstream of the target sites should contain PAM sequence-NGG, and the target sites are assembled into tRNA-sgRNA respectively The BbsI, BsaI, BsmBI and BfuAI loci of the multi-site expression cassette obtained tRNA-sgRNA multi-site expression cassettes targeting the BoMS1 gene and SRK3 gene, respectively, and finally the tRNA-sgRNA multi-site expression boxes targeting the BoMS1 gene and SRK3 gene were obtained. The expression cassette was loaded into an expression vector expressing Cas9 protein to obtain a plant expression vector capable of knocking out BoMS1 gene and SRK gene at the same time. Male sterile line.

The above-mentioned method for creating a male sterile line compatible with the maintenance line at the flowering stage is to use the CRISPR/Cas9 gene editing system to mutate the cabbage BoMS1 gene and the cabbage SRK3 gene. The CRISPR/Cas9 gene editing technology described in the present invention is widely used in crops at present. The gene mutation technology used, its principle and technical route can be obtained through the website, the Internet or other professional journals.

Beneficial effects of the present invention:

1. The present invention provides the key gene BoMS1 gene for controlling male sterility of cabbage, and down-regulates the expression of the cabbage BoMS1 gene by RNAi technology, and then creates a transgenic male sterile line, thus overcoming the use of male sterility approach to carry out the first generation of cabbage hybrids. Breeding of varieties relies heavily on a single ogura CMS single sterile resource, and there is a problem of genetic vulnerability risk.

2. The present invention uses RNAi technology to simultaneously down-regulate the expression of the cabbage BoMS1 gene and the cabbage SRK gene, and creates a transgenic male sterile line that is compatible with the maintainer line during flowering, and realizes the hybridization between the cabbage sterile line and the maintainer line through insect media. For example, bee pollination reduces the reproductive cost of sterile lines, the sterile line created by this method avoids the problem that artificial bud stage pollination is required for the propagation of maintainer lines, and reduces the reproductive cost of male sterile lines of cabbage.

3. The present invention can also create a non-transgenic male sterile line compatible with the maintainer line at the flowering stage by simultaneously mutating the cabbage BoMS1 gene and the cabbage SRK gene.

Description of drawings

Figure 1 is a schematic diagram of the plasmid structure related to the construction of BoMS1 gene interference expression vector, A: PBI525 vector; B: iBoMS1 gene expression cassette; C: PCA13Bar plant expression vector; D: iBoMS1 gene plant expression vector.

Figure 2 is a comparison of flower organ morphology and pollen development between iBoMS1 transgenic cabbage plants and wild-type non-transgenic plants.

Fig. 3 is the expression of the cabbage BoMS1 gene in the Arabidopsis ms1/ms1 mutant to restore fertility, A: the schematic diagram of the PCA13BAR-NP plant expression vector; B. the schematic diagram of the pCA13BarAPMS1-BoMS1 plant expression vector; C: the mutant expressing the BoMS1 gene to restore the fertility .

Figure 4 shows that the PCABARiMS1/iSRK transgenic cabbage plants show complete male sterility and are compatible with the maintainer line during flowering. A: PCABARiMS1/iSRK expression vector; B: PCABARiMS1/iSRK transgenic plants showing male sterility; C: male sterility Cabbage plants were cross-compatible with wild-type plants at flowering. C1-C2: wild-type plants were selfed at bud stage and flowering stage, C3-C4: sterile plants had cross-compatibility with wild-type plants at bud-stage and flowering stages; D: sterile plants had cross-compatibility with wild-type plants at flowering stage.

Figure 5 is the double knockout design of BoMS1 gene and SRK3 gene using CRISPR/Cas9 system. A: BoMS1 gene and SRK3 gene knockout target site; B: tRNA processing-based multiple sgRNAs expression cassette sequences, red letters indicate the AtU6-26 promoter; green letters indicate Arabidopsis glycine-tRNA sequences; blue letters indicate BbsI, BsaI, BsmBI, and BfuAI restriction sites; yellow letters indicate the optimized sgRNA structural sequence, orange letters indicate the terminator sequence; C: schematic diagram of PCACas-tMS1/tSRK double gene knockout plant expression vector, Bar selection marker gene Expression cassettes are not indicated in the figure.

Figure 6 is the mutation detection of BoMS1 gene and SRK3 gene mediated by CRISPR/Cas9-tRNA system, A: BoMS1 gene mutation detection; B: SRK3 gene mutation detection.

Figure 7 shows that BoMS1 knockout plants exhibit complete male sterility. A-D: wild-type control plants (respectively showing the development of flowers, stamens, anthers, and pollen); E-H: BoMS1 knockout plants (respectively showing the development of flowers, stamens, anthers, and pollen).

Figure 8 is the cross-compatibility of BoMS1 and SRK3 double knockout sterile lines with non-transgenic maintainer lines at flowering. A, C, E, G: Wild-type control plant flowering period fluorescence and flowering hybrid affinity knot detection; B, D, F, H: Double gene knockout plants flowering period fluorescence and flowering hybrid affinity knot detection.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments. The following examples will help those skilled in the field of biotechnology breeding to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that, for those of ordinary skill in the field, without departing from the concept of the present invention, several modifications and improvements can also be made in the field of cruciferous plants, but these modifications and improvements belong to the present invention scope of protection.

Unless otherwise specified, the experimental methods used in the following examples are regarded as conventional methods or can be obtained from published experimental instructions or experimental technology websites. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1: Identification of cabbage BoMS1 gene sequence

Combined with bioinformatics analysis, the Brassica Database online database was used to obtain the cabbage gene Bol035718 (BoMS1) sequence (SEQ ID No. 2), and then the genome sequence (including introns, SEQ ID No. 3) of the gene was obtained. According to Bol035718 Gene sequence information, designed primers MS1-F: 5-CATGCCATGGCCATGTCGAATCTGATTCG-3 and MS1-R: 5-CGCGGATCCTCAAGGCAAAAAAGAGAGAGGAATAAG-3. (1) Extracting the total RNA at the bud stage of the self-incompatible line F416 of cabbage, and reverse transcribing it into cDNA; (2) Extracting the genomic DNA of the self-incompatible line F416 of cabbage. Using the extracted cDNA and genomic DNA as templates, the PCR products were cloned and sequenced using MS1-F and MS1-R primers, respectively. The results showed that the cloned target gene coding frame sequence and genomic sequence were both consistent with the Bol035718 gene coding frame sequence in the Brassica Database. completely consistent with the genome sequence.

Example 2: RNAi down-regulates BoMS1 gene expression to obtain male sterile material of cabbage

Using primers PMS1-F (5-AACTGCAGCCTTAAGTATACGACTAGAAACAT-3) and PMS-R (5-TGCCATGGATCGTATCGAACTTTAGTTTGGTC-3), the promoter sequence PBoMS1 (SEQ ID No. 5) of 2068 bp upstream of BoMS1 gene was amplified from cabbage genomic DNA. After sequencing, the sequence was excised with PstI+NcoI and incorporated into the PstI and NcoI sites of the PBI525 vector (FIG. 1-A) to replace the 35S-35S promoter therein. Using primers MS1-F (5-CCATGGCCATGTCGAATCTGATTCGAACAGATC-3) and iMS-2 (5-CGGGATCCCTGTAATGTTCCATAAATAAATGC-3) to amplify the 674bp first exon to first intron sequence, and insert NcoI+ downstream of the PBoMS1 promoter after sequencing BamHI site. In addition, primers iMS-3 (5-CGGGATCCATGTCGAATCTGATTCGAACAGAT-3) and iMS-4 (5-CGGGATCCCAACATATTGGCAATGGTTGCAAC-3) were used to obtain the reverse complementary sequence of the first exon, sequenced and inserted into the BamHI site of the vector obtained in the previous step. After identifying the insertion direction, the BoMS1 gene RNAi hairpin structure (iBoMS1, SEQ ID No. 5) was obtained, and the iBoMS1 expression cassette was obtained (Fig. 1-B). Finally, the iBoMS1 gene expression cassette was excised with PstI+EcoRI and inserted into the PCA13Bar plant expression vector to obtain the iBoMS1 gene interference expression vector shown in Figure 1-C. The plant expression vector shown in Figure 1-C was introduced into the cabbage F416 self-incompatible line by Agrobacterium EHA105-mediated hypocotyl transformation. Positive transgenic plants were obtained by callus induction, adventitious bud induction, adventitious bud rooting and PCR identification on MS medium containing 6 mg/L glufosinate herbicide PPT. The morphological characteristics of the transgenic cabbage plants in the vegetative growth stage were completely consistent with those of the non-transgenic plants, but after flowering, the stamen filaments became shorter, the anthers became hypertrophic and no normal pollen was produced, showing complete male sterility, but its pistil development was not affected. (figure 2).

Example 3: Restoring fertility by expressing BoMS1 gene in Arabidopsis ms1/ms1 male sterile mutant plants

The CS20 mutant (Germplasm/Stock: CS75) in the ABRC Arabidopsis mutant library is an MS1 gene mutant whose homozygous mutant ms1/ms1 exhibits complete male sterility. The 2981 bp MS1 gene upstream promoter APMS1 (SEQ ID No. 6) was amplified from Arabidopsis DNA with primers APMS-F (5-AACTGCAGGAGACCCTCTCTCATCTTGCGTGT-3) and APMS-R (5-AACTGCAGCGAATCAGAAATTTGGTTTGATCTTG-3). Primers MS1-TF (5-CGGGATCCATCATTCATGTTATATTAAACCTTC-3) and MS1-TR (5-GAAGATCTACTACACACTTCTTTCTCCGCCTTG-3) were designed to amplify the AT-MS1 terminator (SEQ ID No. 7) 346 bp downstream of the MS1 gene from Arabidopsis DNA. First, the cloned Arabidopsis thaliana MS1 gene terminator At-MS1 was cut with BamH+BglII and inserted into the BamH site upstream of the Nos terminator in the PCA13BAR-NP plant vector as shown in Figure 3-A, and the desired direction was screened ; The cloned BoMS1 gene coding frame was cut with NcoI+BamHI and inserted between the NcoI and BamHI sites upstream of the At-MS1 terminator; finally the Arabidopsis APMS1 promoter was cut with PstI and inserted into the PstI upstream of the BoMS1 gene site, screen the positive clones in the target direction, and obtain the BoMS1 gene plant expression vector pCA13BarAPMS1-BoMS1 as shown in Figure 3-B. Arabidopsis thaliana MS1 gene mutant CS20 was transformed by inflorescence infection mediated by Agrobacterium GV3101. The harvested seeds were sown at 4°C for 1 week and then sprayed with 500ppm herbicide PPT for about 15 days to screen transgenic positive plants. Among the herbicide-resistant PPT-positive plants, primers MS1-GF (5-CATTTCGTGATCCCCTCAAAGG-3) and MS1-GR (5-GAGGATTGGAGTTTCTCGGTTAAG-3) were used to amplify a 632 bp Arabidopsis thaliana MS1 gene fragment. The genotype identification of the corresponding individual plants was carried out by digestion with the Dicer RsaI. If the PCR product of the corresponding single plant has only a 632 bp band and no band is excised, it indicates that the corresponding genotype of the single plant is ms1/ms1. The fertility of individual ms1/ms1 plants after flowering was further identified, and it was found that complete fertility recovery occurred in some ms1/ms1 genotype plants (Fig. 3-C). It was proved that expressing BoMS1 gene of cabbage in Arabidopsis ms1/ms1 mutant plants could restore its sterile phenotype, which further proved that BoMS1 gene is closely related to the fertility regulation of cabbage.

Example 4: Using RNAi to interfere with BoMS1 and SRK genes at the same time to create a transgenic sterile line of cabbage that is compatible with the flowering period of the maintainer line

An RNAi hairpin structure targeting SRK3 gene interference (iSRK, SEQ ID No. 8) was made by using a sequence of the first exon of SRK3 gene in the self-incompatible line F416 and the Intron sequence from the FLC gene of Brassica juncea , and the RNAi hairpin structure was fused with the SLG13 stigma-specific promoter sequence (SEQ ID No. 9) from cabbage to make the SRK3 gene RNAi interference expression cassette. The SRK3 gene RNAi interference expression cassette was excised with PstI and inserted into the PstI site of the BoMS1 gene RNAi interference plant expression vector shown in Figure 1-D to obtain a plant expression vector PCABARiMS1/iSRK that can interfere with the expression of both BoMS1 and SRK genes at the same time. (Figure 4-A). The plant expression vector was transformed into the cabbage self-incompatible line F416 by Agrobacterium-mediated transformation of cabbage hypocotyls. The obtained 21 transgenic cabbage plants showed little difference from the wild type in the vegetative stage, of which 20 transgenic plants showed complete male sterility. The flowers of the completely sterile plants have shriveled anthers, and are slightly thinner than the wild type, with normal filaments and no pollen. The sterile anther sections were identified by paraffin section, and compared with the fertile plants, the tapetum of the sterile plants was degraded and abnormally expanded, the callus wall outside the free microspores was not degraded, and most of the microspores were vacuolated (Figure 4- B). The non-transgenic cabbage self-incompatibility line F416 was used as the male parent to cross with transgenic sterile plants at the flowering stage, and the cross-compatibility of pollen on the stigma of the transgenic plants was determined. The results showed that the transgenic sterile cabbage plants were pollinated at the bud and flowering stages. Afterwards, a large number of pollen tubes were elongated, and the hybrid compatibility at flowering stage was significantly higher than that of non-transgenic plants (Fig. 4-C). The investigation of the pod-bearing and seed-bearing situation of the hybridization between male sterile plants and corresponding non-transgenic plants at flowering period showed that the transgenic sterile plants, as female parent and corresponding fertile maintainer line, could bear normal seeds at flowering (Fig. 4-D).

Example 5: Simultaneous knockout of BoMS1 gene and SRK gene to create a sterile line of cabbage that is cross-compatible with the maintainer line during flowering

According to the BoMS1 genome sequence shown in SEQ ID No.3, find a site in the second exon and the third exon respectively; find two sites in the first exon for CRISPR/Cas9 system mediation Site-directed mutagenesis of the BoMS1 gene. The CRISPR/Cas9 system-mediated site-directed mutagenesis of the SRK gene was performed at four sites in the first exon of the SRK3 gene of the self-incompatible line F416 (Fig. 5-A). Two complementary primers were synthesized for each targeting site, and the four pairs of primers targeting the BoMS1 gene were as follows:

Site A: 5-TGCAGCCTTTCTAAAACTAGAAGG-3, 5-AAACCCTTCTAGTTTTAGAAAGGC-3;

Site B: 5-TGCAGAGTAGCAAAAGGAGAGCCG-3, 5-AAACCGGCTCTCCTTTTGCTACTC-3;

Site C: 5-TGCAGAGGTTAAGAGAGGCTTCGA-3, 5-AAACTCGAAGCCTCTCTTAACCTC-3;

Site D: 5-TGCAAAGTTGGTCCTTTCAGCTCG-3, 5-AAACCGAGCTGAAAGGACCAACTT-3.

The four pairs of primers targeting the SRK3 gene are as follows:

Site A: 5-TGCAGGCAATAATCTTGTCCTCCT-3, 5-AAACAGGAGGACAAGATTATTGCC-3;

Site B: 5-TGCAGTGGCAGAGCTTCTCGCCAA-3, 5-AAACTTGGCGAGAAGCTCTGCCAC-3;

Site C: 5-TGCAGTGGGATCTTTAGAGTGCAT-3, 5-AAACATGCACTCTAAAGATCCCAC-3;

Site D: 5-TGCAAAACCCTATATCCAACTCCAC-3, 5-AAACGTGGAGTTGGATATAGGGTT-3.

The above-mentioned synthetic targeting site complementary primers were inserted into BbsI, BsaI, BsmBI, and BfuAI of the multiple sgRNA sequence expression cassette based on endogenous tRNA processing according to the steps provided online at http://www.genome-engineering.org/crisr/ Four restriction sites (multiple sgRNA sequence expression cassettes are shown in Figure 5-B). Finally, the multi-site sgRNA expression cassettes of the two genes were simultaneously assembled into the PCABar-Cas9 vector expressing Cas9 protein to obtain the PCACas-tMS1/tSRK double gene knockout plant expression vector (Fig. 5-C). It was introduced into the self-incompatible material F416 of cabbage by Agrobacterium-mediated hypocotyl transformation.

A total of 18 Cas9-positive transgenic cabbage plants were obtained, and the PCR direct sequencing method of DNA fragments in the targeted region was used to detect whether the corresponding plants had mutations near the target site (according to the sequencing map whether there was a heavy peak at the target site to determine the corresponding transgenic single plant mutation). The primers BoSRK3-F: 5-GACATTGTCCGACAGAACCTATG-3 and BoSRK3-R: 5-CTTCACTATTATCTGTGAAATTG-3 were used for the amplification of the targeted region of the SRK3 gene. Primers for amplification of the targeted region of the BoMS1 gene: MS1-F: 5-CCATGGCCATGTCGAATCTGATTCGAACAGATC-3 and MS1-R1: 5-GGAAGACGCGAATGGAGATGAAG-3. According to the results of direct PCR sequencing, 6 of the 18 transgenic plants showed a target site peak in the corresponding region of the BoMS1 gene, and 13 plants of the SRK3 gene showed a target site peak in the corresponding region. Among them, 6 strains of BoMS1 gene were accompanied by mutation of SRK3 gene, so the frequency of simultaneous mutation of both genes reached 33.3%. Further single-clonal sequencing results showed that target site mutations occurred in the form of base substitutions, deletions, insertions, etc., without deletion of large DNA fragments (Figure 6).

Compared with wild-type plants, there was no significant difference in the vegetative growth stage of the single plant with double gene mutation at the same time. After flowering, one single plant was found to be completely male sterile, which showed that the filaments were shorter, the style was prominent, and the anthers were completely pollen-free. The anther development process of the completely sterile plant was identified by paraffin section. It can be seen that the epidermis, the inner wall of the drug chamber, and the middle layer cells are all degraded. There are almost no mature pollen grains in the pollen sac, and a small amount of abortive pollen residues deposited on the drug wall. (Figure 7). Further crossed the sterile plant with pollen from the wild plant with the same S-haplotype at bud stage and flowering stage respectively, and found that pollen could germinate at stigma and extend into style, and could produce pods and seeds normally (Fig. 8).

SEQUENCE LISTING

<110> Southwest University

<120> Cabbage BoMS1 gene and its application in creating sterile materials

<130> 1

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 667

<212> PRT

<213> Brassica oleracea

<400> 1

Met Ser Asn Leu Ile Arg Thr Asp Leu Pro Lys Lys Arg Lys Arg Gly

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530 535 540

Arg Val Phe Arg Glu Ile Tyr Trp Glu Leu Arg Asp Val Val Val Asp

545 550 555 560

Asn Gln Arg Glu Met Ala Pro Arg Val Asp Glu Met Ala Leu Asn Gly

565 570 575

Asn Lys Gly Leu Val Leu Asp Gly Asn Val Gly Val Met Met Asn Ile

580 585 590

Glu Val Met Lys Tyr Tyr Asp Asp Glu Asp Ser Lys Lys Lys Asp Lys

595 600 605

Arg Ile Glu Cys Glu Cys Gly Ala Lys Glu Glu Asp Gly Glu Arg Met

610 615 620

Val Cys Cys Asp Ile Cys Glu Val Trp Gln His Thr Arg Cys Val Gly

625 630 635 640

Val Gln His Asn Glu Glu Val Pro Arg Ile Phe Leu Cys Gln Ser Cys

645 650 655

Asp Gln His Leu Ile Pro Leu Ser Phe Leu Pro

660 665

<210> 2

<211> 2004

<212> DNA

<213> Brassica oleracea

<400> 2

atgtcgaatc tgattcgaac agatcttcca aagaagagga agagaggaga aagtacggtt 60

tttaggctga agactttcgg agagacagga catcccgctg agctgaacca gttgtctttt 120

agagacaacc ttgggaagct tcttgagttt ggtcactttg agagctccgg tctcatggga 180

agttggtcct ttcagctcga ggttcatcgt cacccaaatc ctctatatgt tcttctcttt 240

gtcgtcgaag agcccatcga agcctctctt aacctccgtt gcaaccattg ccaatatgtt 300

ggttgggggaa accacatgat atgcaacaag aagtaccatt tcgtgatccc ctcaaaggaa 360

acaatggccg cctttctaaa actagaagga ggaatcttgg tttctcccga aaaagaaagc 420

ctctcccatc ttgtggaact tcaaggccat gtccttcacg gctttttcca ctccaacgga 480

tttggtcact tgctctcaat caatggcatc gagtccggtt ccgacttaac cggtcatcaa 540

gtcatggagt tgtgggatcg cctctgctct ggtttaaagg ccaggaaaat agggttgaac 600

gatgcatcgc acaagaaagg gatggaactg aggctgctgc atggagtagc aaaaggagag 660

ccgtggttcg gtcgttgggg ttaccggttc gggtcaggga catacggagt gactcaaaag 720

atttacgaga aggcacttga gtcggtccgc aacgtaccct tgtgtttgct taaccatcac 780

ctaaccagcc ttaaccgtga gactccaatc ctcttgtcaa ggtatcaaac tttatccacc 840

gagccattga ttactctcag tgatctcttc atgttcatgc ttcatctcca ttcgcgtctt 900

ccaagagaca actacatgaa taactcccga aaccaaatca tctccattga tagtaccaac 960

tgtagatggt ctcaaaaacg gattcaaatg gctattaaag tggttataga gtctttgaaa 1020

agggtcgaat gccggtggat atcgagacaa gaagtgaggg acgcagctag aaattacatc 1080

ggggacacgg gtttgcttga cttcgtgttg aagtcgctcg gtaaccaagt ggtcgggaac 1140

tatttggtcc gacgtagtct aaacccggtg aagaaagtgc tagagtattg cttggaggac 1200

atatcaaatt tattaccaag tagtaaccat gaactcacaa cccttcaaaa tcaatatcaa 1260

atggggaaga ctactaccac gacgaacgga cataacaaga tcacaagagg tcaagtgatg 1320

aaagacatgg tatactttta caaacacatt cttatggact acaagggagt gttaggcccc 1380

ataggcatat tgaaccaaat cggaatggct tcaagggcta tcctcgacgc taagtacttc 1440

atcaaagagt atcactacat cagagataca ttgatgaaaa caattcagtt agatggaggt 1500

ggcaaattag gaatattctg cacaatcgcg tggaaatctc atcatcataa caacgacata 1560

aagatgcctc cacaagaatg catagtggtg aacaaaaatg caacaatgag tgaagtgtat 1620

agagaggcag agagagtgtt cagagagatc tattgggaac taagagacgt cgtagtggat 1680

aatcaaaggg agatggctcc aagggtcgat gaaatggcat tgaatgggaa caaaggattg 1740

gtgttagatg ggaatgtagg agtgatgatg aacattgaag tgatgaagta ttatgatgat 1800

gaagatagta agaagaagga taagaggatc gagtgtgaat gtggagcgaa ggaggaagat 1860

ggagagagga tggtgtgttg tgatatttgt gaagtatggc aacacacaag gtgtgttggt 1920

gttcaacata acgaggaagt tcctcgcatt tttctttgtc aaagctgtga tcaacatctt 1980

attcctctct ctttttttgcc ttga 2004

<210> 3

<211> 2775

<212> DNA

<213> Brassica oleracea

<400> 3

atgtcgaatc tgattcgaac agatcttcca aagaagagga agagaggaga aagtacggtt 60

tttaggctga agactttcgg agagacagga catcccgctg agctgaacca gttgtctttt 120

agagacaacc ttgggaagct tcttgagttt ggtcactttg agagctccgg tctcatggga 180

agttggtcct ttcagctcga ggttcatcgt cacccaaatc ctctatatgt tcttctcttt 240

gtcgtcgaag agcccatcga agcctctctt aacctccgtt gcaaccattg ccaatatgtt 300

ggtgacgtca tctccctctt tctctccact tctctcattg tctatatata ttttgcatat 360

cgatatctat ctatctatat gtgtgtgtgc atgctgatac atccataata tgatcaacga 420

tcacaacgta acaataaaaa gacctgataa gataatttag ctcaccgagt aagcattttt 480

cgttgcatat ctatgaaaaa tataacaagc ggaaaaaaaa atgatatggt gatccaagag 540

tgttggaggc tcaacctctt ttagaaggat gatagtttca ttgattcaaa tttcatatgt 600

taattttaat atgttacatg taataggtca acaatatgta ttatgcattt atttatggaa 660

cattacaggt tggggaaacc acatgatatg caacaagaag taccatttcg tgatcccctc 720

aaaggaaaca atggccgcct ttctaaaact agaaggagga atcttggttt ctcccgaaaa 780

agaaagcctc tcccatcttg tggaacttca aggccatgtc cttcacggct ttttccactc 840

caacggattt ggtcacttgc tctcaatcaa tggcatcgag tccggttccg acttaaccgg 900

tcatcaagtc atggagttgt gggatcgcct ctgctctggt ttaaaggcca ggtatacgta 960

ttaagattat aattatcatc ttttatattt gtatacgttg tgcaatgttc acgtgataca 1020

tatcatatgg tactaaacca ttcagcagaa ttctatgact tgtcataaat aataaagttt 1080

ggatttttaa ccatttaatt catggtagtc aagtgctttg aagattcaac acatggttga 1140

gagcaatttc aaccatacta gtaactttgt taactcaaga aaccaccact ttatagaact 1200

tgggacgaaa ttctggattt tagtttcgaa ttatagatat tattcgaatt ttctcttttg 1260

cttttagtta taaatttcac atattaggcc attttattag aaatggtaac aattatttgt 1320

atcttattat gttatggtat atatgttgga tataggaaaa tagggttgaa cgatgcatcg 1380

cacaagaaag ggatggaact gaggctgctg catggagtag caaaaggaga gccgtggttc 1440

ggtcgttggg gttaccggtt cgggtcaggg acatacggag tgactcaaaa gatttacgag 1500

aaggcacttg agtcggtccg caacgtaccc ttgtgtttgc ttaaccatca cctaaccagc 1560

cttaaccgtg agactccaat cctcttgtca aggtatcaaa ctttatccac cgagccattg 1620

attactctca gtgatctctt catgttcatg cttcatctcc attcgcgtct tccaagagac 1680

aactacatga ataactcccg aaaccaaatc atctccattg atagtaccaa ctgtagatgg 1740

tctcaaaaac ggattcaaat ggctattaaa gtggttatag agtctttgaa aagggtcgaa 1800

tgccggtgga tatcgagaca agaagtgagg gacgcagcta gaaattacat cggggacacg 1860

ggtttgcttg acttcgtgtt gaagtcgctc ggtaaccaag tggtcgggaa ctatttggtc 1920

cgacgtagtc taaacccggt gaagaaagtg ctagagtatt gcttggagga catatcaaat 1980

ttattaccaa gtagtaacca tgaactcaca acccttcaaa atcaatatca aatggggaag 2040

actactacca cgacgaacgg acataacaag atcacaagag gtcaagtgat gaaagacatg 2100

gtatactttt acaaacacat tcttatggac tacaagggag tgttaggccc cataggcata 2160

ttgaaccaaa tcggaatggc ttcaagggct atcctcgacg ctaagtactt catcaaagag 2220

tatcactaca tcagagatac attgatgaaa acaattcagt tagatggagg tggcaaatta 2280

ggaatattct gcacaatcgc gtggaaatct catcatcata acaacgacat aaagatgcct 2340

ccacaagaat gcatagtggt gaacaaaaat gcaacaatga gtgaagtgta tagagaggca 2400

gagagagtgt tcagagagat ctattgggaa ctaagagacg tcgtagtgga taatcaaagg 2460

gagatggctc caagggtcga tgaaatggca ttgaatggga acaaaggatt ggtgttagat 2520

gggaatgtag gagtgatgat gaacattgaa gtgatgaagt attatgatga tgaagatagt 2580

aagaagaagg ataagaggat cgagtgtgaa tgtggagcga aggaggaaga tggagagagg 2640

atggtgtgtt gtgatatttg tgaagtatgg caacacacaa ggtgtgttgg tgttcaacat 2700

aacgaggaag ttcctcgcat ttttctttgt caaagctgtg atcaacatct tattcctctc 2760

tctttttttgc cttga 2775

<210> 4

<211> 987

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 4

ccatggatgt cgaatctgat tcgaacagat cttccaaaga agaggaagag aggagaaagt 60

acggttttta ggctgaagac tttcggagag acaggacatc ccgctgagct gaaccagttg 120

tcttttagag acaaccttgg gaagcttctt gagtttggtc actttgagag ctccggtctc 180

atgggaagtt ggtcctttca gctcgaggtt catcgtcacc caaatcctct atatgttctt 240

ctctttgtcg tcgaagagcc catcgaagcc tctcttaacc tccgttgcaa ccattgccaa 300

tatgttggtg acgtcatctc cctctttctc tccacttctc tcattgtcta tatatatttt 360

gcatatcgat atctatctat ctatatgtgt gtgtgcatgc tgatacatcc ataatatgat 420

caacgatcac aacgtaacaa taaaaagacc tgataagata atttagctca ccgagtaagc 480

atttttcgtt gcatatctat gaaaaatata acaagcggaa aaaaaaatga tatggtgatc 540

caagagtgtt ggaggctcaa cctcttttag aaggatgata gtttcattga ttcaaatttc 600

atatgttaat tttaatatgt tacatgtaat aggtcaacaa tatgtattat gcatttattt 660

atggaacatt acagggatcc caacatattg gcaatggttg caacggaggt taagagaggc 720

ttcgatgggc tcttcgacga caaagagaag aacatataga ggatttgggt gacgatgaac 780

ctcgagctga aaggaccaac ttcccatgag accggagctc tcaaagtgac caaactcaag 840

aagcttccca aggttgtctc taaaagacaa ctggttcagc tcagcgggat gtcctgtctc 900

tccgaaagtc ttcagcctaa aaaccgtact ttctcctctc ttcctcttct ttggaagatc 960

tgttcgaatc agattcgaca tggatcc 987

<210> 5

<211> 2068

<212> DNA

<213> Artificial

<220>

<223> Artificial

<400> 5

ccttaagtat acgactagaa acattaaaat gacatgtatc agttttgtgt taaaaaaaag 60

agacatgtat cagtttgatg gttgatttga aagctttcaa aaccatatgt aagataaaag 120

tcaaaataat ttaactgtga aaacaatact gttcactttt tcaagaatgt gttcgaggaa 180

aaaaataaga ttttttatgtc ataatttgtt taatgtacac tagagaacat tatattaacc 240

taaaatataa gaagtgtgta ttctttcaca aaataaaaat agctacgaaa ttacctaata 300

atatttacat atatatggta attaatgatt atgaataata aagatttgat aacaattttt 360

gcatccttct tcatttttgt ttaaatttat attattaaaa aaacttaaac aatcacatta 420

accatataat aaaaaattag atttttttctt atatgttata ttttgaattt ttttaaatga 480

ctttaaatta caaaaatgag gaaaccttat atgttttttt taaacgactt taaaatacaa 540

aaattaggac accttatatg ttatattttt cttatatgtt agaattttct tatatgttat 600

attttgaatt ttttaaaacg actttaaatt acaaaaatgt aagttttcct taagtatacg 660

actaaaaaca ttaaaatgac atgtatcaat tcattggttg atttgaaagc tttcaaaacc 720

atatggaaga taaaagtcaa aataattcaa ctgtgaaaac aatactgttc atttttttca 780

agaatgtgtt ctatggaaaa ataaggtttt tatgtcataa tttgtttaat gttcaatccg 840

atcaatccca tgatgtatta attatagttt tgttccatta ttttttaataa aaattgatcc 900

gatccatcgg aaagaaatta tataataaca acaaaaatat tttatatata taaataaaat 960

gatcaaatat ataaaagaaa ctaccaaaat atatacaaat aaattcaccc tgcgtaagtc 1020

ttatcctagt atattagtgt tttcgaatac tttctttatc catattgttt cgtgtggtca 1080

ccaatcgata cttgtatttt agacacggtc tcttttgttg tttagccctc ccaaagtttt 1140

agtgggtttt atgattttga taaagcccaa ttaaaaagat tcctatgatc aagaggtata 1200

taggctttat ttgttttctt ttattttttg gtcaaaggct ttatttgttt tttagtcgcc 1260

acaaattaca ttcatataca tcaccaatca ctcagtacaa acctcatttg atgagactga 1320

gaatcgatta aagtttgaag tccgcagcta cgctatgctg catgcttgca tatgaaacga 1380

aatctgatat atagactagc gtcaatatac acacgtgtgt ttgcgtgtac atatagaaat 1440

aatagataca tgtaagtgca ggtagctcat gacagaaatt gacggaacac agagttactc 1500

tgattctata aaatagtggg aagctcaaat gcatgtcttc attattttat tattatatct 1560

tctcatgttc tcaagccacg tgacattcct caccctcgat ctttttgcat gcgtatacac 1620

acaaatacat atataggatg catatacaat aactaatggc ggatactaat taaattatca 1680

gtcctttcaa ataaatattt gtatgaaaga tagatttggg acagtggggt tcgaatgttt 1740

tgtatttaat tattctatct aagaaagaac agttgttaat ggttgtaaga atacaagagt 1800

catgcacata tcaagaccta acatccaatc cattagtatc aattgttaaa tcacagttta 1860

attacccttt ctctttctct cactcacttt tccttttcac cataaagcac taatcgtgtg 1920

atcatcatct taatgactac tatatctcta aacccttcac acacacgtcc tcaacacttc 1980

tcggcctctt ataattataa caaacccatg atatataaat ccaaaccaac cagtatctct 2040

tgtcaagacc aaactaaagt tcgatacg 2068

<210> 6

<211> 2981

<212> DNA

<213> Arabidopsis thaliana

<400> 6

gagaccctct ctcatcttgc gtgtatcttc tcctctatct atatatatat aacagccact 60

aacatgcaga catcaaacta tctctcttta aatctcaaga acaagaacta aactactact 120

agtcttcacc aaggtaaagc tactttggaa cccgagaacc aagttaattt gttctcatcg 180

aatttttttt gttctcatat gttgaaattt tgttattgtc atcttcactt tttgattgat 240

cgatagaaaa ataagtattc aattgatcaa ttctttttat atatatacat gttttataaa 300

ccatggatct ccatctaaag ttttgtttta ctgtgtttct tattctgatt cttgtagaat 360

aatatatcaa cacacatagt atactcttct tggaaggcaa ggaacatgaa tcaagaagct 420

gcgagtaacc ttggacttga tctgaaacta aatatttcac cgtcgttgga ttcatctttg 480

ctcactgaga gttcatcgtc atcgttatgc tcggaggaag ctgagggtgg aggaggag 540

gctaaatcaa tggtggtcgt tggctgccct aattgcatca tgtacatcat cacttcttta 600

gagaatgacc ctagatgccc tagatgcaac agccatgttt tgcttgattt tctcacagga 660

aaccatagca agaagagcac tagttaaaaa aaaaaataaa aaaaaaatac ataatattaa 720

attaaggatg ttcttttatt tttctttctt ttttcttttc cttaatttgt tgccacattt 780

attgatagtt tgaattaatc cacaatgaac ggatatgttc ataaaatcga aacctatata 840

cttatgtttt taaaacatcc ttaacttcat ttgtaatatt gactgcttaa cgatggataa 900

ttcttgtaga aaactaagca atattaatat tgattctttg caacgggtga ttacaaaaat 960

ttaaacttgg attaatagct agtataaatg atggatgtgt cattgtgtcc tcatcaaaag 1020

gttacgtatt cggactattg cgatatttaa ttttataaat ggaaaatggt cgttgttgac 1080

ttgttataag ttcaatgtat atgtacatgg cacaattaat tatgttttta atatgaaaaa 1140

tgtggaacaa tgacttttttt ttcttttcct ttttttgcac tatgtaattc tagcgttaaa 1200

tgcgaagtat atacataaat atatacacat catacgtcaa acaataaata ggtttggttt 1260

acttgttacc aaagttgttg aactaacaaa ttaaatgttt ataaaaacat gaaaaagagt 1320

tatgcttatt ataaatgtga aaaatcttca cctaaactta ctataaaaaa aatgcatgtt 1380

ggttttacat taatgtcgga ttcaacatta cgtgtgtttt gttcctttcc ccaactatca 1440

aaagaaacaa cacgtatcct ttaagtagat ttctcacatt tttatcatta tatttaccgc 1500

tactggcaat ttcaattgga caaaccaaaa ccaaatatat attttttcgc tgacgtaaga 1560

cctatcaaag aaatctatca ttcaaccaac aactataaaa acgaatcaaa gtcacttgtt 1620

ggctactatt caactaacaa tataatgtat cgctgcctcg ctggtcgttg ttacttgtta 1680

ctaagaatcg tgaaacaaaa atgatacaaa attatcaaca attattcagt ctacgtacgt 1740

atatacctac ttatactatt agtggctgtc aaagtttaaa ctttaaacac taaacaagtg 1800

atagtaggtc aacattttag ttagatatgc ttaatattga ttcaaactca aatattgcaa 1860

ttgttagcat cgggatttaa caaggcatat cattaatttt aaaaaaagat taaaatttga 1920

ttggtctgaa cttttaactc tttttttttta cgaagattgt aacactaaaa acatatattt 1980

gaagcactga agtgactgta agtacccaaa gttgttgtga tcactgcaat cattaaggtg 2040

agtacaattt tgaatgtaat atattaacta ttaagtaaat accattgcaa tttgcaaacc 2100

actttttctg agaacataat ttgaaacttt ctttaaaccg ctaaaaaaca tctattgatg 2160

taaacacctt tttctattga tgtaacatat taattatgtt taattaacaa taactatgtg 2220

ggtttgaaca tatagtgatg acaaatcatc aacactatca caaatcaata aactacatat 2280

caattaaaac attgataatc gactaaattg agtttgtcca accacgcaaa cacacacgtg 2340

tgtgttatgt aatagatgca tatatgtgca ggtagacatg acagaaattg acggaacaca 2400

gagttaatct gattctttaa aaatgtgaga agctcaaatg catgtcttca ttattttatt 2460

atattatctt ctcatgttcc caagtcacgt gacattcctc accctcgatc tttttgcatg 2520

ccatttgtca catattctta cacaaataca tgtatatgta ttaaaattat caatccagtt 2580

tcgaatatta cagaagtatt tttagaaatg aaattagatt tgggaataag aagggtttga 2640

atgttttttt gaaaaatagt taattatttg gctaagaaag aaaagttgtt aatgttacaa 2700

gtcatgcaca tatcaagacc taacatacaa tccaatggta acaattgtta attcacagtt 2760

taattaacct ttctctttct ctcactcact tttccttttc atcaccacaa agcactaatt 2820

gtgtgaccat catcttaatg actactatat ctctaaaccc ttaacacact tcctcaacac 2880

ttttcggtct cttcttataa caaaccccat cataatatat atccaaatct atcatattcc 2940

tattccctct cttctcaaga tcaaaccaaa tttctgattc g 2981

<210> 7

<211> 346

<212> DNA

<213> Unknown

<220>

<223> Unknown

<400> 7

atcattcatg ttatattaaa ccttcttttt ttctatcatt acgtcataat acataaatta 60

tactcctcga tcaacgtgaa ttctaatgat ccctcctgcg tctatatcac caacatgttg 120

aatcacaaag tcatatcctt gcctcctctg gagcctgact gttctaataa tatcattatc 180

aacatgtcgc tctcctcttc ccctgatgac tcatgtgacg tcatctactc caaccgtgat 240

tccaagtttt acttgacaac aaccttaagg cttgtctcca agaaaaggca gttaaatttt 300

aacagaaaat aattcacaac acaaggcgga gaaagaagtg tgtagt 346

<210> 8

<211> 1186

<212> DNA

<213> Artificial

<220>

<223> Artificial

<400> 8

ccatgggcaa tagaacactt gtatctcccg gtgatgtctt cgagctcggt ttcttcaaaa 60

ccacctcaag ttctcgttgg tatctcggta tatggtacaa gacattgtcc gacagaacct 120

atgtatggat tgccaacaga gataacccta tatccaactc cactggaacc ctcaaaatct 180

caggcaataa tcttgtcctc cttggtgact ccaataaacc tgtttggtcg acgaatctaa 240

ctagaagaag tgagagatct ccagtggtgg cagagcttct cgccaacgga aacttcgtga 300

tgcgagactc caacaacaac gatgcaagtc aatttttatg gcaaagtttc gattacccta 360

cagatacttt gcttcctgac atgaaactgg gttacgacct aaaaacaggg ctggacagat 420

tccttacatc atggagaagt ttagatgatc cgtcaagcgg gaatttctcg tacaccatgg 480

gttagatata tgatttgata gcacttcaga tatcttctct tgtggtgaga gcctcaaaat 540

acttgtgtta gtttctctaa gtgtgcttta tgagcttgca agtctacagc aaacttcttc 600

ataacgcatc ttatctatca ggaatgatgc aagtgttttg aataccagaa tctgaaatag 660

gtttagaaac ttggtaactg atgaacatgt tgtccgtatt aaacagggat cctgtacgag 720

aaattcccgc ttgacggatc atctaaactt ctccatgatg taaggaatct gtccagccct 780

gtttttaggt cgtaacccag tttcatgtca ggaagcaaag tatctgtagg gtaatcgaaa 840

ctttgccata aaaattgact tgcatcgttg ttgttggagt ctcgcatcac gaagtttccg 900

ttggcgagaa gctctgccac cactggagat ctctcacttc ttctagttag attcgtcgac 960

caaacaggtt tattggagtc accaaggagg acaagattat tgcctgagat tttgagggtt 1020

ccagtggagt tggatatagg gttatctctg ttggcaatcc atacataggt tctgtcggac 1080

aatgtcttgt accatatacc gagataccaa cgagaacttg aggtggtttt gaagaaaccg 1140

agctcgaaga catcaccggg agatacaagt gttctattgc ggatcc 1186

<210> 9

<211> 411

<212> DNA

<213> Artificial

<220>

<223> Artificial

<400> 9

taaccaattt acgttatacc aaatttttca accctctttt tagtaaaaaa cgaaattaaa 60

gttttttccc tcttagtccg acgattttaa gctaattagt tcgaacaaag agtacaacat 120

taattttcta acagacttag atgcacttgc gaacaacata cttgctgaac accatatgtt 180

atgttggcag ggtgagaaat taatcacgtg tagatataga agtagtagac aaatgatata 240

ggtttgtggg aatgaattaa tcgtagggat gaaaaagtca tcgaacatgt aacaccacat 300

tttacttgtc tgctaggttc gtgatagtcg tttaaattag atacgtgaaa aaagattata 360

aatatgcaaa aggggaaggg gaagaaaaga aagaaaaagg aggggagaga a 411

Claims (5)

1. Down-regulated or mutated cabbageBoMS1Use of the expression of genes for the creation of male sterile plant lines, characterized in that said cabbage isBoMS1The amino acid sequence of the gene coding protein is shown as SEQ ID No.1, and the cabbage isBoMS1The nucleotide sequence of the gene is shown as SEQ ID No.2 or SEQ ID No. 3; the application is to be inserted withiBoMS1The plant expression vector of the gene expression cassette is transferred into the cabbage by agrobacterium transfection cabbage hypocotyl transformation method to obtain transgenic male sterile line, and the transgenic male sterile line is obtainediBoMS1The nucleotide sequence of (A) is shown as SEQ ID No. 4;iBoMS1has a promoter inserted upstream ofPBoMS1，PBoMS1The nucleotide sequence of (A) is shown as SEQ ID No. 5.

2. Down-regulated or mutated cabbageBoMS1Use of gene expression for creating a male sterile line with cross-compatibility with the flowering phase of a maintainer line, characterised in that the cabbage is selected from the group consisting ofBoMS1The amino acid sequence of the encoded protein of the gene is shown in SEQ ID No.1, the cabbageBoMS1The nucleotide sequence of the gene is shown as SEQ ID No.2 or SEQ ID No. 3; the application is to be inserted withiBoMS1The plant expression vector of the gene expression cassette is transferred into the cabbage by agrobacterium transfection cabbage hypocotyl transformation method to obtain transgenic male sterile line, and the transgenic male sterile line is obtainediBoMS1The nucleotide sequence of (A) is shown as SEQ ID No. 4;iBoMS1has a promoter inserted upstream ofPBoMS1，PBoMS1The nucleotide sequence of (A) is shown as SEQ ID No. 5; the plant expression vector is also inserted withiSRK3A gene expression cassette, saidiSRK3The gene expression cassette containsiSRK3，iSRK3The nucleotide sequence of (A) is shown as SEQ ID No. 8.

3. Use according to claim 2, characterized in that saidiSRK3Is inserted upstream ofSLG13A promoter, a promoter-specific protein,SLG13the nucleotide sequence of the promoter is shown as SEQ ID No. 9.

4. The use according to claim 2 or 3, wherein the plant expression vector of claim 2 or 3 is transformed into Brassica oleracea by Agrobacterium transfection of the hypocotyl of Brassica oleracea to obtain a transgenic male sterile line with cross-compatibility with the flowering phase of the maintainer line.

5. Down-regulated or mutated cabbageBoMS1Use of the expression of genes for the creation of male sterile plant lines, characterized in that said cabbage isBoMS1The amino acid sequence of the encoded protein of the gene is shown in SEQ ID No.1, the cabbageBoMS1The nucleotide sequence of the gene is shown as SEQ ID No.2 or SEQ ID No. 3; according to the sequence characteristic requirements of CRISPR/Cas9 gene editing system target sitesBoMS1Gene exons andSRK34 target sites with the length of 20bp are respectively selected from the gene exons, and the downstream of the target sites contains a PAM sequence-NGG; to is directed atBoMS1The nucleotide sequence of the target site containing PAM sequence-NGG of the gene exon is 5'-GCCTTTCTAAAACTAGAAGGAGG-3', 5'-GAGTAGCAAAAGGAGAGCCGTGG-3', 5'-CCATCGAAGCCTCTCTTAACCTC-3' and 5'-AAGTTGGTCCTTTCAGCTCGAGG-3'; for the purpose ofSRK3The nucleotide sequence of the target site containing PAM sequence-NGG of the gene exon is 5'-GTGGCAGAGCTTCTCGCCAACGG-3', 5'-AACCCTATATCCAACTCCACTGG-3', 5'-GTGGGATCTTTAGAGTGCATCGG-3' and 5'-GGCAATAATCTTGTCCTCCTTGG-3'; respectively assembling the target sites to BbsI, BsaI, BsmBI and BfuAI sites of tRNA-sgRNA multi-site expression cassettes to obtain targetsBoMS1Genes andSRK3tRNA-sgRNA multi-site expression cassette of gene is packaged, and finally target is carried outBoMS1Genes andSRK3the tRNA-sgRNA multi-site expression cassette of the gene is loaded into an expression vector for expressing Cas9 protein, so that the genes can be knocked out simultaneouslyBoMS1Genes andSRKthe plant expression vector of the gene is used for transforming the cabbage self-incompatible line to breed the non-transgenic male sterile line which is compatible with the maintainer line in the flowering phase in a hybrid way.

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