CN109879945B

CN109879945B - Function and application of brassica napus pod dehiscence resistance gene BnIND

Info

Publication number: CN109879945B
Application number: CN201910218648.9A
Authority: CN
Inventors: 范楚川; 周永明; 翟云孤; 蔡胜利
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-03-21
Filing date: 2019-03-21
Publication date: 2021-05-28
Anticipated expiration: 2039-03-21
Also published as: CN109879945A

Abstract

The invention belongs to the technical field of rape molecular breeding, and discloses a function and application of a cabbage type rape pod shatter resistance gene BnIND, wherein two homologous copies of the gene have partial redundant functions in the pod shattering process of rape, and the function of the BnA03.IND copy in the pod shattering process is larger than that of the BnC03.IND copy; sequence comparison analysis finds that two copies of the BnIND gene code proteins with the same function, and the promoter region has larger sequence variation; the sequence difference of the promoter regions causes the difference of the gene expression quantity of the two copies of the BnIND gene, and finally leads the BnA03.IND copy to play a larger role in the pod dehiscence process of rape than the BnC03.IND copy. The invention provides important functional genes and breeding materials for the genetic improvement of the pod shattering resistance of the rape.

Description

Function and application of brassica napus pod dehiscence resistance gene BnIND

Technical Field

The invention belongs to the field of rape molecular breeding, and relates to a function and application of a cabbage type rape gene BnIND in the aspect of pod crack resistance.

Background

Dehiscence of mature siliques is a biological property of oilseed rape which develops during long-term evolution in order to adapt to the environment and propagate progeny. However, the easy cracking property of the siliques is a disadvantageous property in rape production, can cause reduction of the yield of the rape, and is not beneficial to popularization and application of mechanized harvesting. Statistically, the resulting yield loss per year typically accounts for about 20% of the total yield, up to 50% under severe weather conditions (Price et al, 1996; Child et al, 1998). Therefore, developing an anti-dehiscent oilseed rape variety suitable for mechanized harvesting would bring significant economic benefits.

In crucifer (Brassicaceae) plants, including Arabidopsis and oilseed rape, dehiscence of the silique is caused by the development of specialized cells into a delamination zone between two fruit lobes. The separation layer area consists of a lignification layer and a separation layer; during the ripening of the siliques, the lignified layer hardens and the separating layer secretes hydrolytic enzymes causing a reduction in its resistance to cracking, the combined action of which causes the siliques to crack from the separating layer.

In Arabidopsis, there has also been a great deal of research on the genes associated with dehiscence of siliques and their regulatory networks. Wherein two functionally redundant MADS-box genes SHATERPROOF 1(SHP1) and SHP2 are involved in the development of the valve margins (Liljegren et al, 2000); they regulate the expression of the downstream INDEHISCENT(IND) gene. The loss-of-function mutants of the genes can not form normal fruit flap edges, so that the siliques can not crack and seeds can not scatter. The pod development of the brassica napus is very similar to that of arabidopsis thaliana, so that the research on the anti-cracking mechanism in arabidopsis thaliana provides rich information for the related research in the brassica napus.

Brassica napus is an allotetraploid species, which is naturally hybridized and postformed from two diploid ancestral species, cabbage (b.rapa, AA genome) and brassica oleracea (b.oleracea, CC genome) (Chalhoub et al, 2014). The siliques of Brassica napus are susceptible to dehiscence, and conventional methods have introduced resistance to resistance genes in closely related species, mainly by interspecific crossing (Prakash and Chotra 1988; Morgan et al, 1998; Summers et al, 2003).

In summary, the problems of the prior art are as follows:

the methods of the prior art are time consuming and also introduce other undesirable agronomic traits, making them unsuitable for cultivation applications (Summers et al, 2003). Compared with interspecific hybridization, the anti-crack gene in arabidopsis thaliana can be quickly and effectively introduced into a good variety through genetic transformation, and has been successfully applied to genetic improvement of silique cracking in brassica crops (Chandler et al, 2005;

etc., 2006), but this approach is currently limited by transgenic policy in many countries and regions. In addition to interspecies crosses and transgenes, corner resistance can be improved by inducing new genetic variations. For example, Braatz et al (2018) obtained anti-keratotic mutants by EMS mutagenesis and demonstrated increased resistance to keratosis in mutants with only two copies of ind, indicating that the two copies of the gene have functionally conserved and redundant propertiesAnd (5) carrying out characterization. However, random mutagenesis also results in a large number of background mutations and adversely affects other traits. Both randomly mutagenized single mutants, such as ind, have very significantly reduced fertility (Braatz et al, 2018). Another major drawback of random mutagenesis in Brassica napus is that its polyploid nature results in multiple functionally similar homologous copies of most genes, and random mutagenesis usually mutates only one of them randomly and thus does not generally produce a significant phenotype.

The significance of solving the technical problems is as follows:

in recent years, CRISPR/Cas9 has been successfully applied to rape as an efficient site-directed gene editing technology. The invention utilizes CRISPR/Cas9 technology to target BnIND homologous genes, obtains mutant single plants through genetic transformation, obtains single-copy homozygous and double-copy homozygous mutants through multi-generation selfing separation, and performs phenotypic identification, genetic analysis and dehiscence resistance measurement on the mutants. The results of the study indicate that double-copy mutants of the BnIND gene and homozygous mutants with a single bna03.ind copy mutation can produce siliques with significantly increased dehiscence resistance. The BnIND gene has huge application potential and prospect for improving the dehiscence character of rape pod, and provides a new gene resource for the yield breeding of rape.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the function and the application of the cabbage type rape BnIND gene in the aspect of pod crack resistance, and simultaneously provides the function of identifying two homologous copies of the cabbage type rape BnIND gene in the aspect of pod crack resistance by using a gene editing technology. The invention targets two homologous copies of the cabbage type rape BnIND gene, quickly and efficiently obtains the cabbage type rape germplasm resource with stable inheritance and obvious crack resistance angle phenotype, and has important significance for the breeding of the crack resistance cabbage type rape. The mutant does not contain T-DNA insertion, and compared with the wild type, the anti-cracking angle index reaches a very significant level.

The invention is realized in such a way that the nucleotide sequence of the candidate gene BnIND for the pod shatter resistance of the cabbage type rape is SEQ ID NO: 12. SEQ ID NO: 13.

the invention also aims to provide a protein coded by the brassica napus dehiscence resistance gene BnIND, and the amino acid sequence of the protein is as follows: SEQ ID NO: 24. SEQ ID NO: 25.

the other purpose of the invention is to provide a promoter for controlling the anticracking angle of the brassica napus by separating the anticracking angle gene BnIND of the brassica napus, wherein the nucleotide sequence of the sequence promoter is SEQ ID NO: 26. SEQ ID NO: 27.

the invention also aims to provide a double mutant for carrying out mutation by using the brassica napus anti-dehiscence gene BnIND, wherein the double mutant simultaneously generates mutation of nucleotide sequences in gene coding regions from two copies of the BnIND, a plant containing the mutation has a dehiscence shape, and the nucleotide sequences of the double mutant generating the dehiscence phenotype after mutation are as follows: SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22 and SEQ ID NO: 23.

another object of the present invention is to provide a single mutation using the brassica napus dehiscence resistance gene BnIND, wherein the single mutation is a mutation of a nucleotide sequence within a gene coding region from a single copy of BnIND, a plant containing the mutated nucleotide sequence has a dehiscence resistance shape, and the single mutated nucleotide sequence generating the dehiscence resistance phenotype after mutation is SEQ ID NO: 28.

the invention also aims to provide an application of the double mutation in breeding.

Another object of the present invention is to provide a method for breeding plants using said single mutation.

The invention also aims to provide an application of the brassica napus dehiscence resistance gene BnIND in the improved brassica napus dehiscence resistance seeds.

In the invention, the nucleotide sequence table of the candidate gene BnIND for the pod shattering resistance of the cabbage type rape is as follows:

>BnA03.IND

ATGTCTGGCTCAAAAGCAGATGCAGCCATAGCCCCAATAGTCATGATGGAGCATCATCATCTCCTTATGAATTGGAACAAACCTATTGATCTCATTACAGAAGAAAACTCTTTTAACCACAATCCTCATTTCATAGTAGATCCACCTTCCGAAACCCTAAGCCACTTCCAGCCCCCGCCGACAATCTTCTCCGATCACGGAGGAGGAGAGGAAGCAGAAGAAGAAGAAGAAGAAGAAGGAGAGGAAGAGATGGATCCGATGAAGAAGATGCAATACGCGATTGCTGCCATGCAGCCCGTAGACCTCGATCCAGCCACCGTTCCTAAGCCGAACCGCCGTAACGTAAGGGTAAGCGACGACCCTCAGACGGTGGTGGCTCGTCGGCGTAGAGAAAGGATAAGCGAGAAGATCCGGATATTGAAGAGGATGGTGCCAGGCGGTGCAAAGATGGACACTGCCTCCATGCTCGACGAAGCCATCCGCTACACCAAGTTCTTGAAACGGCAGGTGAGGCTAGCTTCTTCAGCCTCACACTCAGCTTGGAGCTCCTATGTCTGA SEQ ID NO：12.

>BnC03.IND

ATGTCTGGTTCAAAAGCAGATGCAGCAGCCATAGCTCCAATAGTCATGATGGAGCCTCATCATCTCCTTATGAACTGGAACAAACCTATTGATCTCATTACACAAGAAAACTCTTTTAACCACAATCCTCATTTCATGGTAGATCCACCTTCCGAAACCCTAAGCCACTTCCAGCCCCCGCCGACAGTCTTCTCCGATCACGGAGGAGGAGAGGAAGCAGAAGACGAAGAAGGAGAGGAAGAGATGGATGAGATGAAGGAGATGCAATACGCGATTGCTGCCATGCAGCCCGTAGACATCGATCCAGCCACCGTTCCTAAGCCGAACCGCCGTAACGTAAGGGTAAGCGAGGACCCCCAGACGGTGGTGGCTCGTCGGCGTAGAGAAAGGATAAGCGAGAAGATCCGGATATTGAAGAGGATGGTGCCAGGCGGTGCAAAGATGGACACTGCCTCCATGCTCGACGAAGCCATCCGCTACACCAAGTTCTTGAAACGGCAGGTGAGGCTTCTTCAGCCTCACACTCAGCTTGGGGCTCCTATGTCTGACCCTTCTTGCCTTTGTTATTACCACAACTCGGATACCTAA SEQ ID NO：13.

amino acid sequences of proteins such as

>BnA03.IND

MSGSKADAAIAPIVMMEHHHLLMNWNKPIDLITEENSFNHNPHFIVDPPSETLSHFQPPPTIFSDHGGGEEAEEEEEEEGEEEMDPMKKMQYAIAAMQPVDLDPATVPKPNRRNVRVSDDPQTVVARRRRERISEKIRILKRMVPGGAKMDTASMLDEAIRYTKFLKRQVRLASSASHSAWSSYV SEQ ID NO：24。

>BnC03.IND

MSGSKADAAAIAPIVMMEPHHLLMNWNKPIDLITQENSFNHNPHFMVDPPSETLSHFQPPPTVFSDHGGGEEAEDEEGEEEMDEMKEMQYAIAAMQPVDIDPATVPKPNRRNVRVSEDPQTVVARRRRERISEKIRILKRMVPGGAKMDTASMLDEAIRYTKFLKRQVRLLQPHTQLGAPMSDPSCLCYYHNSDT SEQ ID NO：25。

The promoter sequence is:

>BnA03.IND

GGGTGAGGTATCTCCATTTCAATTCTTCTCTTTATATATTAATCGAATTATTTACGTATGAAATGAACGTTTATATAGAAATTTCGTGTGGAAAACGACATGTACACGGCATCTCAAGACCAATTAGTAATATACTTTAGTGGTGATTACATGTTTACTTATCCAATTGAGAATTTAAAGCATCGACAATACCTTAATGTCGATTAAGCCGTCCCCACTTCATGTAATGAGTTATGGGGGGAGAGAGAGATCCCGAAATTCGTCAAATAAAACAACTTAGAACTAAAAACCGACACCAAGTATCATAAAGGAAATGTTGAAGAAGTCATTTATCGTATCCAGCTCACAATTCCTAAGATTAAATCATGACCGTTGGAAGAGCTTATAAGATTAAACTGAAGAAATTGTGGGTTTTAGAAGAAAGACAAGAAAGAGAAGAACATGATCTTACATTGCCTATTTTGGTGTATAGGAGTTGTCAAAAAGAGGAGAGAGAGGAGACAATTAGGTCAAATAAATGAGCACTAAAAATGGAGACATGTGTTGAGTAACTATTACAAGAGCGACTTATGCTTCTATATGGCAATGATATCATCACCAAAGTGCAATGCCCCTTTTTGCCCTAGTTTCGTAAAGTCTCTCTCCTTCTTCGTCCTTAGGAAAAACCCTAAATTAAATCCTGTGTTCTTGATCTTTCTTTTTGAGTAACCATGATTTTGACCACACACTAGTTCTTCTATATTTTGTGGTCTATAGGATTTTGCTTTATATGTGTTTCTTGTATTGCTCCGTACGTGCGTATATAAATTTAAATGGTTACAACAAGGTTTATTATAAATAGGCACAAATTAGTCCATGAAGTTATTTAGCTTGCACAAGTATAATTTGTTAAGTATTTAAATATATAAATTTGTTACAAAACTTAATTAAATTTATCTGATTATATTTTCTTTAGTGTTCTTCCTTTGCCAACGTTGAGGTAGCTATTATTATTATTATTTTGAACATTATGTACGTAGTTATCTTGGCTAGTTATGATTCGAATTCTTAATTTGGATCACACTTAACAGTATTTAAAATATTCTTAGAACTAAAATAATTAAGAGTTACCTTTAAATTGAAGTATTCGTGCTAAACAGAAACTAGAATAAACAAATGATTGCATGTTAATTTTTTTTTTCGATTTTCCTATCAGAATAAACACATGATTGCATGCAAATTTTGTTTTTGATTACGTTATCTTTTGTTTATTTTAGTTTTGATGCTAATTAATATTTTTTATTAACAACTCACATACATTCTACCTGATTCTAGGTCAGATAATGACACAGCGCAACAAAATTAATACAAAACCTTCGGAAAGTAGAATACCGCAGAAGTAACTTTTTTGGGTACATACGAAATACAGTGAAATCTCTATAAATTAATAATGTTGGGACTATACCAAAACTATAATTTTTTATTAATTTATAGAGATTAATTTATCGATATACTAATTGAATCAAAAACTTAATTTGAGACTAAAAAATTATATTATTTTATAGAGATTTTTAGTGTATATTAATTTATAGAATATTATTTTATAAAAAATTTTAGTGTGTATTAATTTATAGAGTATTAATTTAAAGAGGTTATACTGTAATGTGAATCTTCGAAAAACATGCCATACATAACCACGGATCATAGTCGACACCTCAACGTGAAGCAAATTTGACAATTTACATACATAACCAACAAAAAGTAGAATACCTTGAAAATTTAAAACCCAAAATATGATGTAAAACTCAAGCTTGGTCCAGAGCATAAAAAAATTAAAGCCATCGCTTTGGTATCACATATTTAAACGTCAGTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGGGGGGGTAATATAAAAATATAATTAACAAAAAAAAATTATGAAACAATTAGCATGTAAAACACTAATCTTTTGGTTGTGACAAAACGTTTTCACAAATGTTCTATAAATAAATTCAAGTGCATTTTATCTGCAAAATATATACTTTCACTCATAAAATAAGAGCGTTTAAAACATTCATACACGCACTACATTGACATGACAAAAGAAATCCGCAAATACACATGATGTATGTCGAAAAAAACAAAAAATACACATGATGTATATATAGAGAGGATAGTATCTAGGAAATAAGACTATATTATATATATAAAGAAAATAGAGAAAAGATAAAAATATAAATTGGTATGTATAAAAGAAAGGTCTATGCGTCTCTAG SEQ ID NO：26。

>BnC03.IND

CTAGAGACGCATAGACCTTTCTTTTATACATACCAAAATTTTTTTCTCTAATTTCTTTATATATATAATATAGTCTTATTTCCTAGATATATCCGAACTAAATATGTTTGTATTTGCGGATTTCTTTTGTCATGTCAATCTAGTGCGTATATGAATGTTTTAAACGCTCTTATTTTATGAGTGAAAGTATATATTTTGCAGATAAAATGTGCTTGAATTTATTTATAGAACATTTGTGAAAACGTTTTGTCACAACCAAAAGACTAGTGTTTTACATGCTAATTATTTCATAAATTTTCTTGATATTTATATTTTTATATTACTTCCCCAAAAAAAAAAACTGACGTTTAAATATCTGATACCAAAGCGATGGCTTTAATTTTTTTATGCTCTGGACCAAGCTTGAGTTTTACATCATATTTTGGGTTTAGGTTTTCACGGTATTCTACTTTTTGTTGGTTATGTATGTAGATTGTCAAATTTGCTTCACGTTGAGGTGTCGACTATGATCCGTGGTTATGTCGTATGGCATGATTTTTGAAGATTCAAACTACTTCGTATGTCTACCCAAAAATGTTACTTCCGCGGTAATCTACTTTCCGAAAGTTTTGTATAATTTTGTTGCGCTGTGTCATTATCTGACCTAGAATCAGGTAGAATGTATGGAAGTTGTTAATAAAAAAATATTAATTAGCATCAAAACTAAAATAAACAAAAGATAACATAATCAAAAACAAAATTTGCATGCAATCATGTGTTTATTCTGATATATAGGATATTCGAAAAAAAAAATAACATGCAATCATTTGTTTATTGTAGTTTCTGTTTAACACGAATACTTCAATTTCAAGTTAACTCTTAATTATTTTAGTACTAAGAATATTTTAAATAGTATTTTTTTAAATACTGTTAAGTGTTATCCAAATTAAGAATTTGAATCATAACTAGCCAAAATAACTACGTACATAATACATAATGTTCAAAATAATAATAATAATAATAATAATAATAATAATAATAGCTACCTCAACTTTGGCAAATGAAGAACACTAAAGAAAATATAATCAGATAAAGTTAATCAAGTTTTGTAGCAAATTTATATATTTAAATACTTAACACACACACACACACACACATTTATATACCTCTTGTGCAAGCTAAATAACCTCATGGACTAATTTGTGCCTGTTTATAATAAACCTTAATTGTTGTAACCATTTAAATTTATATACGCACGTACGG SEQ ID NO：27。

nucleotide sequences of double mutants, e.g.

IND-59-14-8 aaccS4

CCATCC-GCTACACCAAGTTCTTG wt

CCATCCAGCTACACCAAGTTCTTG +A

CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：14。

IND-67-3-2 aacc S4

CCATCC-GCTACACCAAGTTCTTG wt

CCA----GCTACACCAAGTTCTTG -3bp

CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：15。

IND-146-1-1 aacc S4

CCATCC-GCTACACCAAGTTCTTG wt

CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：16。

S1

CCAATA-GTCATGATGGAGCCTCA wt

CCAATATGTCATGATGGAGCCTCA +T SEQ ID NO：17。

IND-105-1-6 aacc

S4

CCATCC-GCTACACCAAGTTCTTG wt

CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：18。

S1

CCAATA-GTCATGATGGAGCCTCA wt

CCAATATGTCATGATGGAGCCTCA +T SEQ ID NO：19。

S3

AGCCGAACCGCCGTAACGTAAGG wt

AGCCGAAC--------CGTAAGG -8bp SEQ ID NO：20。

S4

CCATCC-GCTACACCAAGTTCTTG wt

CCATCCAGCTACACCAAGTTCTTG +A.SEQ ID NO：21。

IND-210-2-2 aacc S4

CCATCC-GCTACACCAAGTTCTTG wt

CCATCCTGCTACACCAAGTTCTTG +T SEQ ID NO：22。

S4

CCATCCGCTACACCAAGTTCTTG wt

GCTACACCAAGTTCTTG -17bp.SEQ ID NO：23。

Single mutant nucleotide sequences such as

IND-201-4-10 aaCC S4

CCATCCGCT---ACACCAAGTTCTTG wt

TGGACACCAAGTTCTTG -11bp,+3bp SEQ ID NO：28。

In the invention, by cloning the genome sequence of the anticracking gene BnIND of the Brassica napus and utilizing the CRISPR/CAS9 technology to target two homologous copies of the BnIND, a mutant is obtained, and the obtained mutant single strain is as follows:

IND-5-6-4 AAcc、IND-230-4-9 AAcc、IND-201-4-10 aaCC、IND-59-14-8 aacc、IND-67-3-2 aacc、IND-146-1-1 aacc、IND-105-1-6 aacc、IND-210-2-2 aacc。

the invention finds that the anti-crack angle indexes of the double-copy homozygous mutant of the BnIND gene and the homozygous mutation of a single BnA03.IND copy are remarkably increased by measuring the anti-crack angle indexes of the horns of the obtained mutant and a wild single plant.

According to the invention, through phenotype and microstructure observation of the obtained mutant and the wild type silique, the double homozygous mutant and the homozygous mutant with single BnA03.IND copy mutation have different tissue structure differences in the structure of the edge of the fruit flap compared with the wild type. Sequence comparison analysis finds that two copies of the BnIND gene code proteins with the same function, and the promoter region has larger sequence variation; further gene expression analysis proves that the expression quantity of the BnA03.IND copy in the development process is obviously higher than that of the BnC03.IND copy. Therefore, it can be seen that the difference in gene expression amount between the two copies of the BnIND gene is caused by the sequence difference of the promoter regions, and finally the BnA03.IND copy plays a greater role in the pod dehiscence process of rape than the BnC03.IND copy.

In summary, the advantages and positive effects of the invention are:

the CRISPR/Cas9 used in the invention is an efficient fixed-point targeted gene editing technology, the technology is strong in pertinence, and the mutant can be obtained quickly and efficiently by knocking out the genes of rape with the CRISPR/Cas 9. The mutant is planted and is subjected to multi-generation selfing separation, so that the homozygous mutant without the T-DNA insertion can be obtained. The method is faster than the traditional crossbreeding technology and safer than mutation breeding.

Microscopic observation of the obtained mutant siliques revealed that most mesocarp cells of the mutant of BnIND have thicker cell walls than the wild type at the endocarp border. In the double mutant, there is almost no lignified layer and separating layer at the edge of the pericarp, and the b-layer of lignified endocarp is directly connected with the lignified vascular bundle of the septum, forming a continuous lignified mass around the pericarp to prevent dehiscence of the horn. The lignification structure of the single mutants, although similar to the wild type, still has some minor differences. In contrast to the wild type, the single mutants exhibited a small layer of non-lignified cells forming a separate layer. In addition, in the bna03.ind mutant, the pericarp and diaphragm are tightly connected together by lignification bridges to prevent dehiscence of the horn. However, in both wild type and bnc03.ind single mutants, a distinct separation layer was present between the lignified endothelial layer and the lignified septal vascular tissue.

The experiment obtains the germplasm resources of the brassica napus mutant with the characteristic of crack resistance, and compared with the wild type, the mutant crack resistance index is remarkably increased. The material provides valuable resources for rape breeding programs.

The gene of the invention is an important gene for controlling pod dehiscence in cabbage type rape, two homologous copies of the gene have partial redundant functions in the pod dehiscence process of the rape, and the function of BnA03.IND copy in the pod dehiscence process is larger than that of BnC03.IND copy; sequence comparison analysis finds that two copies of the BnIND gene code proteins with the same function, and the promoter region has larger sequence variation; further gene expression analysis proves that the expression quantity of the BnA03.IND copy in the process of horn development is obviously higher than that of the BnC03.IND copy; the sequence difference of the promoter regions causes the difference of the gene expression quantity of the two copies of the BnIND gene, and finally leads the BnA03.IND copy to play a larger role in the pod dehiscence process of rape than the BnC03.IND copy. The invention provides important functional genes and breeding materials for the genetic improvement of the pod shattering resistance of the rape.

Drawings

Fig. 1 is a flowchart of a method for obtaining mutants of brassica napus resistant to pod shattering by using CRISPR/CAS9 technology, provided by an embodiment of the present invention.

FIG. 2 is a gene structure diagram and a vector diagram of two copies of BnIND provided in the examples of the present invention.

In the figure: (1) the white box indicates that the gene contains one exon, the grey area indicates the bHLH domain of the gene, the vertical line in the gene model indicates the target site, and the arrow indicates the direction of the sgRNA. The target sequences, underlined PAM regions, are shown at S1-S4. (2) Construction of the SBnIND vector.

FIG. 3 is a graph of gene expression in different tissues in J9707 according to an embodiment of the present invention.

Fig. 4 is a measurement result of the anti-crack angle coefficient provided by the embodiment of the present invention, which shows that the anti-crack angle coefficient of the bna03.ind pure mutant and the double homozygous mutant is significantly higher than that of the wild type.

FIG. 5 is a phenotypic graph showing wild type and mutant provided in the examples of the present invention.

FIG. 6 is a microscopic view of a section of a silique at stage 18 of its development as provided by an embodiment of the present invention.

In the figure, a, b, c and d are structural schematic diagrams before phloroglucinol is dyed; e, f, g and h are effect graphs after phloroglucinol staining of paraffin sections, and the silique fruit flap edge development of the BnIND gene double homozygous mutant and the BnA03.IND copy simple homozygous mutant is abnormal.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the prior art, a CRISPR/Cas9 technology is not utilized to target a homologous gene of BnIND, a mutant single plant is obtained through genetic transformation, and a double-copy homozygous mutant is obtained through multi-generation selfing separation. The mechanism of the angle resistance of the mutants with loss of BnIND function was not elucidated and the reason for the technical effect of the two copies on the angle resistance was not resolved.

To solve the above problems, the present invention will be described in detail with reference to specific embodiments.

The nucleotide sequence of the candidate gene BnIND for the anticracking horn of the brassica napus provided by the embodiment of the invention is SEQ ID NO: 12. SEQ ID NO: 13.

the embodiment of the invention provides a protein coded by the anticracking gene BnIND of Brassica napus, and the amino acid sequence of the protein is as follows: SEQ ID NO: 23. SEQ ID NO: 24.

the embodiment of the invention provides a promoter for separating the cabbage type rape anti-crack-angle gene BnIND and controlling the cabbage type rape anti-crack angle, wherein the nucleotide sequence of the sequence promoter is SEQ ID NO: 25. SEQ ID NO: 26.

the embodiment of the invention provides a double mutant for carrying out mutation by using the cabbage type rape anti-dehiscence gene BnIND, the double mutant simultaneously generates the mutation of a nucleotide sequence in a gene coding region by two copies of the BnIND, a plant containing the mutation has the dehiscence shape, and the nucleotide sequence of the double mutant generating the anti-dehiscence phenotype after the mutation is as follows: SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22 and SEQ ID NO: 23.

the embodiment of the invention provides a single mutation for carrying out mutation by using the cabbage type rape anti-crack angle gene BnIND, wherein the single mutation generates the mutation of a nucleotide sequence in a gene coding region by a single copy of the BnIND, a plant containing the mutated nucleotide sequence has the anti-crack angle shape, and the single mutation nucleotide sequence generating the anti-crack angle phenotype after the mutation is SEQ ID NO: 28.

>BnA03.IND

>BnC03.IND

amino acid sequences of proteins such as

>BnA03.IND

>BnC03.IND

The promoter sequence is:

>BnA03.IND

>BnC03.IND

nucleotide sequences of double mutants, e.g.

IND-59-14-8 aaccS4

CCATCC-GCTACACCAAGTTCTTG wt

CCATCCAGCTACACCAAGTTCTTG +A

CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：14。

IND-67-3-2 aacc S4

CCATCC-GCTACACCAAGTTCTTG wt

CCA----GCTACACCAAGTTCTTG -3bp

CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：15。

IND-146-1-1 aacc S4

CCATCC-GCTACACCAAGTTCTTG wt

CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：16。

S1

CCAATA-GTCATGATGGAGCCTCA wt

CCAATATGTCATGATGGAGCCTCA +T SEQ ID NO：17。

IND-105-1-6 aacc

S4

CCATCC-GCTACACCAAGTTCTTG wt

CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：18。

S1

CCAATA-GTCATGATGGAGCCTCA wt

CCAATATGTCATGATGGAGCCTCA +T SEQ ID NO：19。

S3

AGCCGAACCGCCGTAACGTAAGG wt

AGCCGAAC--------CGTAAGG -8bp SEQ ID NO：20。

S4

CCATCC-GCTACACCAAGTTCTTG wt

CCATCCAGCTACACCAAGTTCTTG +A.SEQ ID NO：21。

IND-210-2-2 aacc S4

CCATCC-GCTACACCAAGTTCTTG wt

CCATCCTGCTACACCAAGTTCTTG +T SEQ ID NO：22。

S4

CCATCCGCTACACCAAGTTCTTG wt

GCTACACCAAGTTCTTG -17bp.SEQ ID NO：23。

Single mutant nucleotide sequences such as

IND-201-4-10 aaCC S4

CCATCCGCTACACCAAGTTCTTG

TGGACACCAAGTTCTTG -11bp,+3bp SEQ ID NO：28。

The invention is further described with reference to specific examples.

Examples

The embodiment of the invention provides a method for obtaining a single mutant of an anti-pod brassica napus by using a CRISPR/CAS9 technology, wherein the single mutant of the anti-pod brassica napus is as follows:

IND-5-6-4 AAcc、IND-230-4-9 AAcc、IND-201-4-10 aaCC、IND-59-14-8 aacc、IND-67-3-2 aacc、IND-146-1-1 aacc、IND-105-1-6 aacc、IND-210-2-2 aacc；

as shown in fig. 1, in the method for obtaining an anti-dehiscent brassica napus mutant by using CRISPR/CAS9 technology provided by the embodiment of the present invention, a CRISPR/CAS9 technology is used to target a homologous gene of BnIND, a mutant single plant is obtained by genetic transformation, and a homozygous mutant is obtained by self-crossing separation; and the obtained mutants are subjected to phenotypic identification, angular crack resistance measurement and genetic analysis.

The method specifically comprises the following steps:

s101, vector construction: four sgrnas were designed using CRISPR-P program, the first three sgrnas targeting upstream of the bHLH domain, the targeting sequence of the fourth sgRNA within the bHLH domain. The pYLCRIPSR/Cas9 multi-genome targeting vector system is used for vector construction, and the constructed vector is verified by sequencing.

S102, genetic transformation: genomic DNA and coding sequences for bna03.ind and bnc03.ind were isolated from brassica napus inbred J9707, which are suitable for agrobacterium-mediated transformation. The vector is transferred into a pure line J9707 of semi-winter cabbage type rape by agrobacterium-mediated hypocotyl genetic transformation, and the tissue culture seedlings obtained after hygromycin screening are placed in a greenhouse for growth (light/dark 16/8 hours at 22 ℃).

S103, positive identification: the positive is confirmed by PCR of tissue culture seedlings in the greenhouse, and the presence of T-DNA is verified by PCR amplification using specific primer pairs.

S104, editing and detecting: the mutation of the positive transgenic plant of the T0 generation is preliminarily screened by using a polyacrylamide gel electrophoresis (PAGE) based method, the edited single plant after PAGE gel screening is directly sequenced or cloned into a pEASY-T1 vector by PCR, and the genotype of the transgenic plant is determined by Sanger sequencing.

S105, selfing and homozygosis: the obtained T0 generation editing single plant self-pollinates to generate T1 generation and T2 generation, and double homozygous mutants are obtained by PCR product sequencing near the target site, and all the double homozygous mutants can cause frame shift mutation to generate protein without function. A batch of double homozygous mutants without T-DNA insertion was obtained by PCR verification.

S106, analyzing gene expression quantity: RNA was extracted from different tissues in J9707 and qRT-PCR experiments were performed with CDNA inverted from RNA to detect the expression levels of bna03.ind and bnc03.ind in different tissues.

S107, crack resistance angle measurement: and (3) carrying out crack resistance angle measurement on the obtained mutant and wild type by adopting a random collision method. 20 whole siliques were placed in a cylindrical beaker (internal diameter 13.4cm, height 20.5cm), 13 steel balls of diameter 14 mm were placed in the beaker and shaken on a shaker at 300 rpm for 10 minutes, and the number of cracked siliques in the beaker was recorded every two minutes. According to the formula of crack resistance angle

And calculating the crack resistance angle coefficient.

S108, phenotypic observation: flowers were marked at anthesis and siliques were collected about 5 weeks after anthesis for microscopic examination. The middle of the silique was cut to 5mm length and fixed and embedded in paraffin. A cross-section 8 μm thick was obtained using a Leica RM 2016 microtome. The cross section was analyzed for lignin, and the sections were treated with 2% phloroglucinol for 2 minutes and photographed by color development in 50% hydrochloric acid. Images were obtained using a Nikon ECLIPSE 80i composite microscope. The morphology of the whole mature siliques was observed using a zoom stereomicroscope (SMZ-U, Nikon, Japan) and photographed.

In step S103, the positive identification primer PB-R is:

GCGCGCggtctcTACCGACGCGTATCC SEQ ID NO：1。

BnINDS3-F

attGTGGCTTAGGGTTTCGGAAGG SEQ ID NO：2。

in step S104, the editing and identifying primers are:

BnIND-1：GAAAGGTCTATGCGTCTCTAGTC SEQ ID NO：3。

BnIND-5 AGGAGAGGAAGAGATGGCTCC SEQ ID NO：4。

BnIND-7 CTGAGTGTGAGGCTGAAGAAG SEQ ID NO：5。

BnIND-12 GTGAGGCTGAAGAAGCTAGC SEQ ID NO：6。

BnIND-14 GGAAGAGATGGATGAGATGAACG SEQ ID NO：7。

BnIND-15 AGGGTCAGACATAGGAGGC SEQ ID NO：8。

BnIND-16 TCTTCTTCTGCTTCCTCTCCTC SEQ ID NO：9。

in step S104, the gene cloning primer BnIND-2 GGTCTATGCGTCTCTAGTCCAA SEQ ID NO: 10.

BnIND-17 CAACATGAAACGCGTGATAGAA SEQ ID NO：11。

in the step S106 anti-corner test, the obtained mutants are:

IND-201-4-10 aaCC

S4

CCATCCGCT---ACACCAAGTTCTTG wt

a-----------TGGACACCAAGTTCTTG -11bp,+3bp

IND-5-6-4 AAcc

S1

CCAATA-GTCATGATGGAGCCTCA wt

c CCAATAAGTCATGATGGAGCCTCA +A

IND-230-4-9 AAcc

CCAATAGTCATGATGGAGCCTCA wt

c CCAA--GTCATGATGGAGCCTCA -2bp

IND-59-14-8 aacc

S4

CCATCC-GCTACACCAAGTTCTTG wt

a CCATCCAGCTACACCAAGTTCTTG +A

c CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：14。

IND-67-3-2 aacc

S4

CCATCC-GCTACACCAAGTTCTTG wt

a CCA----GCTACACCAAGTTCTTG -3bp

c CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：15。

IND-146-1-1 aacc

S4

CCATCC-GCTACACCAAGTTCTTG wt

a CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：16。

S1

CCAATA-GTCATGATGGAGCCTCA wt

c CCAATATGTCATGATGGAGCCTCA +T SEQ ID NO：17。

IND-105-1-6 aacc

S4

CCATCC-GCTACACCAAGTTCTTG wt

a CCATCCAGCTACACCAAGTTCTTG +A SEQ ID NO：18。

S1

CCAATA-GTCATGATGGAGCCTCA wt

c CCAATATGTCATGATGGAGCCTCA +T SEQ ID NO：19。

S3

AGCCGAACCGCCGTAACGTAAGG wt

c AGCCGAAC--------CGTAAGG -8bp SEQ ID NO：20。

S4

CCATCC-GCTACACCAAGTTCTTG wt

c CCATCCAGCTACACCAAGTTCTTG +A.SEQ ID NO：21。

IND-210-2-2 aacc

S4

CCATCC-GCTACACCAAGTTCTTG wt

a CCATCCTGCTACACCAAGTTCTTG +T SEQ ID NO：22。

S4

CCATCCGCTACACCAAGTTCTTG wt

C//----GCTACACCAAGTTCTTG -17bp.SEQ ID NO：23。

the nucleotide sequence of the BnIND gene of the genomic DNA separated in the step S102 is as follows:

>BnA03.IND

>BnC03.IND

the invention is further described with reference to specific examples.

Examples

The method for acquiring the pod shatter resistance gene BnIND function of the brassica napus provided by the embodiment of the invention comprises the following steps:

firstly, gene cloning: a semi-winter rape pure line J9707 (seeds from the national engineering research center of Wuhan rape seeds in China) is planted, genome DNA is extracted from fresh and tender leaves, the specific preparation method refers to a method for effectively extracting the total DNA of the rape leaves, the method reported in the university of agriculture in Huazhong, 1994,13(5): 521-. The genomic DNA and coding sequence of BnA03.IND and BnC03.IND are cloned and separated from the extracted DNA.

(II) vector construction: analysis of the genomic DNA and coding sequences of the isolated bna03.ind and bnc03.ind, four sgrnas were designed on two copies of BnIND using CRISPR-P program. The first three sgrnas target upstream of the bHLH domain, and the targeting sequence of the fourth sgRNA is within the bHLH domain. Four sgrnas contained a one-base variation in only the first copy of bna03.ind in both copies, and the other three sgrnas were identical in both copies. Constructing a vector by using a pYLCRIPSR/Cas9 multiple genome targeting vector system, and verifying the constructed vector by sequencing;

and (III) transferring the constructed vector into a pure line J9707 of the semi-winter cabbage type rape by using an agrobacterium-mediated hypocotyl genetic transformation method to perform the following steps:

1) and (3) sterilization:

a. the seeds are put into a small half tube by a 2ml centrifuge tube, 75% alcohol is added to soak the seeds for 3min, the attention time cannot be too long, otherwise, the seeds are not easy to germinate.

b. Removing alcohol, adding 84 disinfectant (diluted by one time with distilled water), and soaking the seeds for 3 min; the sterilizing liquid is removed 84 times, and the container is rinsed 4 to 5 times with sterile water. Note: sterile water with the concentration of 84 disinfectant, namely commercial 84 disinfectant is 1: 1; the amount of seed used is determined by the amount of seed to be killed, and the seed is completely immersed.

2) Sowing: a. sterile seeds were sown to M0 with sterile forceps, 10-12 per dish. b. The culture dish was placed in a sterile culture box and incubated in dark at 24 ℃ for 6 days.

3) Shaking the bacteria: after 4 days of sowing, the target Agrobacterium strain was cultured with LB. Adding 4ml of LB culture solution into a sterilized PU bottle, sucking 10uL of activated target bacteria, and adding antibiotics, namely 4ml of LB liquid, 4uL kan, 4uL Gent and 10uL bacteria night; the strain was shaken in a shaker at 28 ℃ and 180 ℃ and 220rpm for about 15 h. And (3) injecting positive detection of target bacteria before shaking the bacteria. Selecting Agrobacterium strain stored at-80 deg.C, streak-culturing on LB (kanamycin and gentamicin resistant) plate, and dark-culturing at room temperature (about 28 deg.C) for 40-48 hr; picking a single colony and shaking the colony while detecting: a, shaking bacteria: the number and the half spot were picked and inoculated in 1mL of liquid LB containing two antibiotics, cultured at 28 ℃ and 220 r/min. b, detection: and (3) cracking the other half selected spots by using 0.02M NaOH for 10min, carrying out PCR detection by using specific primers, setting negative and positive controls, and selecting positive colonies according to results.

4) Preparation and infection of explants:

measuring OD value (about 0.8 in LB, preferably 16 hr), sucking 2ml of cultured strain into 2ml sterile centrifuge tube, centrifuging at 6000rpm for 3min, and removing supernatant; resuspend once with 2ml DM, centrifuge at 6000rpm for 3min, discard the supernatant, suspend again with 2ml DM, put the suspended bacteria liquid at 4 ℃ for use.

Shearing the hypocotyl of the seedling after sowing for 6 days by using sterile scissors, putting 18ml of M1 liquid culture medium in a sterile plate in advance, wherein the length of the cut explant is 0.8-1.0cm, and vertically shearing the explant as much as possible once when the explant is cut. When each dish contains 150-200 explants, adding 2ml of DM suspension prepared in the first step, starting to dip-stain for 15min (the time cannot be long, but the explants are easy to die), and shaking once every 4-5 times. Then the bacterial liquid is quickly sucked out, the explant is transferred to a sterile plate padded with 3 layers of filter paper, and a large amount of bacterial liquid attached to the explant is sucked away.

5) Transfer to M1 medium, 20-25 explants per dish and deposit in the dark at 24 ℃.

6) After 2-3 days, they were transferred to M2 and incubated in light (24 ℃ day 16 hs/night 8 hs).

7) Transfer to M3 after 3 weeks, and subculture every 2-3 weeks until green shoots appear.

8) Transferred into an M4 rooting culture medium for rooting, and the rooting time is 2-4 weeks. After healthy and complete seedlings are grown, the seedlings can be transferred to a field or a greenhouse for normal growth and fructification.

The tissue culture seedlings obtained after hygromycin screening are placed in a greenhouse for growth (at 22 ℃ under light/dark 16/8 hours);

(IV) detecting the mutant:

(1) positive identification of transgenes is carried out on the mutant individuals by using a specific primer BnINDS3-F/PB-R, and positive individuals containing T-DNA insertion are selected.

(2) According to the sequences near the target, primer premier5 is used to design primers according to the sequences near the target fragment, and blast analysis is carried out after the primers are determined to ensure that no other homologous sequences exist.

(3) PCR amplification was performed using the designed primers for the target fragment.

(4) The PCR amplification effect is detected by electrophoresis on a 1% agarose level.

(5) And detecting the PCR amplification product by a non-denaturing PAGE gel electrophoresis method.

(6) The edited individuals after the PAGE gel screening were PCR amplified and the products were sent directly to the company for genotype determination of transgenic plants using Sanger sequencing.

(V) selfing and homozygosis: the obtained T0 generation editing single plant self-pollination generates T1 generation and T2 generation, single copy homozygous mutant and double copy homozygous mutant are obtained by PCR product sequencing near the target site, and the homozygous mutants can cause frame shift mutation to generate protein with loss of function. Obtaining a batch of double homozygous mutants without T-DNA insertion through PCR sequencing verification;

(VI) analysis of the expression level of the gene: RNA of different tissues in J9707 is extracted, and qRT-PCR experiments are carried out by using cDNA transcribed from the RNA, and the two copies are found to have higher expression level in a nutritional reproductive organ and the highest expression level in the silique peel, and meanwhile, the expression level of BnA03.IND is far higher than that of BnC03. IND.

(VII) determining the crack resistance angle: and (3) carrying out crack resistance angle measurement on the obtained mutant and wild type by adopting a random collision method. 20 whole siliques were placed in a cylindrical beaker (internal diameter 13.4cm, height 20.5cm), 13 steel balls of diameter 14 mm were placed in the beaker and shaken on a shaker at 300 rpm for 10 minutes, and the number of cracked siliques in the beaker was recorded every two minutes. And calculating the crack resistance angle coefficient according to a crack resistance angle calculation formula. As a result, the double homozygous mutant has an increased anti-crack angle coefficient of about 1.3 times compared with the wild type, and has reached a remarkable level.

(eighth), observation of phenotype: flowers were marked at anthesis and siliques were collected about 5 weeks after anthesis for microscopic examination. The results show that the flap edge structure of the double mutant siliques is clearly different from the wild type. The middle of the silique was cut to 5mm length and fixed and embedded in paraffin. A cross-section 8 μm thick was obtained using a Leica RM 2016 microtome. The cross section was analyzed for lignin, and the sections were treated with 2% phloroglucinol for 2 minutes and photographed by color development in 50% hydrochloric acid. Images were obtained using a Nikon ECLIPSE 80i composite microscope. The morphology of the whole mature siliques was observed using a zoom stereomicroscope (SMZ-U, Nikon, Japan) and photographed. Microscopic examination of the obtained mutants and the wild type siliques revealed that most mesocarp cells of the mutants of BnIND have thicker cell walls than the wild type at the endocarp border. In the double mutant, there is almost no lignified layer and separating layer at the edge of the pericarp, and the b-layer of lignified endocarp is directly connected with the lignified vascular bundle of the septum, forming a continuous lignified mass around the pericarp to prevent dehiscence of the horn. The lignification structure of the single mutants, although similar to the wild type, still has some minor differences. In contrast to the wild type, the single mutants exhibited a small layer of non-lignified cells forming a separate layer. In addition, in the bna03.ind mutant, the pericarp and diaphragm are tightly connected together by lignification bridges to prevent dehiscence of the horn. However, in both wild type and bnc03.ind single mutants, there was a distinct separation layer between the lignified endothelial layer and the lignified septal vascular tissue.

The single homozygous mutant and the double homozygous mutant of BnA03.IND have extremely obviously increased crack resistance angle coefficients. Sequence comparison analysis finds that two copies of the BnIND gene code proteins with the same function, and the promoter region has larger sequence variation; further gene expression analysis proves that the expression quantity of the BnA03.IND copy in the process of horn development is obviously higher than that of the BnC03.IND copy. Therefore, it can be seen that the difference in gene expression amount between the two copies of the BnIND gene is caused by the sequence difference of the promoter regions, and finally the BnA03.IND copy plays a greater role in the pod dehiscence process of rape than the BnC03.IND copy.

The invention is further described with reference to specific examples.

Fig. 4 is a result of measuring the anti-crack angle coefficient provided by the embodiment of the present invention, which shows that the anti-crack angle coefficient of the bna03.ind pure mutant and the double homozygous mutant is significantly higher than that of the wild type.

FIG. 6 is a microscopic image of a section of a silique at stage 18 of its development as provided by an embodiment of the present invention.

In the figure, a, b, c and d are structural schematic diagrams before phloroglucinol is dyed; e, f, g and h are the effect graphs after phloroglucinol staining of paraffin sections, and BnIND gene mutation makes the edge of the silique fruit flap abnormally developed.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> function and application of brassica napus pod dehiscence resistance gene BnIND

<130> JZWH2019178

<140> 2019102186489

<141> 2019-03-21

<160> 30

<170> SIPOSequenceListing 1.0

<210> 1

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gcgcgcggtc tctaccgacg cgtatcc 27

<210> 2

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

attgtggctt agggtttcgg aagg 24

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gaaaggtcta tgcgtctcta gtc 23

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aggagaggaa gagatggctc c 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctgagtgtga ggctgaagaa g 21

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtgaggctga agaagctagc 20

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggaagagatg gatgagatga acg 23

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agggtcagac ataggaggc 19

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tcttcttctg cttcctctcc tc 22

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggtctatgcg tctctagtcc aa 22

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

caacatgaaa cgcgtgatag aa 22

<210> 12

<211> 558

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgtctggct caaaagcaga tgcagccata gccccaatag tcatgatgga gcatcatcat 60

ctccttatga attggaacaa acctattgat ctcattacag aagaaaactc ttttaaccac 120

aatcctcatt tcatagtaga tccaccttcc gaaaccctaa gccacttcca gcccccgccg 180

acaatcttct ccgatcacgg aggaggagag gaagcagaag aagaagaaga agaagaagga 240

gaggaagaga tggatccgat gaagaagatg caatacgcga ttgctgccat gcagcccgta 300

gacctcgatc cagccaccgt tcctaagccg aaccgccgta acgtaagggt aagcgacgac 360

cctcagacgg tggtggctcg tcggcgtaga gaaaggataa gcgagaagat ccggatattg 420

aagaggatgg tgccaggcgg tgcaaagatg gacactgcct ccatgctcga cgaagccatc 480

cgctacacca agttcttgaa acggcaggtg aggctagctt cttcagcctc acactcagct 540

tggagctcct atgtctga 558

<210> 13

<211> 588

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgtctggtt caaaagcaga tgcagcagcc atagctccaa tagtcatgat ggagcctcat 60

catctcctta tgaactggaa caaacctatt gatctcatta cacaagaaaa ctcttttaac 120

cacaatcctc atttcatggt agatccacct tccgaaaccc taagccactt ccagcccccg 180

ccgacagtct tctccgatca cggaggagga gaggaagcag aagacgaaga aggagaggaa 240

gagatggatg agatgaagga gatgcaatac gcgattgctg ccatgcagcc cgtagacatc 300

gatccagcca ccgttcctaa gccgaaccgc cgtaacgtaa gggtaagcga ggacccccag 360

acggtggtgg ctcgtcggcg tagagaaagg ataagcgaga agatccggat attgaagagg 420

atggtgccag gcggtgcaaa gatggacact gcctccatgc tcgacgaagc catccgctac 480

accaagttct tgaaacggca ggtgaggctt cttcagcctc acactcagct tggggctcct 540

atgtctgacc cttcttgcct ttgttattac cacaactcgg atacctaa 588

<210> 14

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ccatccgcta caccaagttc ttgccatcca gctacaccaa gttcttgcca tccagctaca 60

ccaagttctt g 71

<210> 15

<211> 67

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ccatccgcta caccaagttc ttgccagcta caccaagttc ttgccatcca gctacaccaa 60

gttcttg 67

<210> 16

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ccatccgcta caccaagttc ttgccatcca gctacaccaa gttcttg 47

<210> 17

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ccaatagtca tgatggagcc tcaccaatat gtcatgatgg agcctca 47

<210> 18

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ccatccgcta caccaagttc ttgccatcca gctacaccaa gttcttg 47

<210> 19

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ccaatagtca tgatggagcc tcaccaatat gtcatgatgg agcctca 47

<210> 20

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

agccgaaccg ccgtaacgta aggagccgaa ccgtaagg 38

<210> 21

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ccatccgcta caccaagttc ttgccatcca gctacaccaa gttcttg 47

<210> 22

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ccatccgcta caccaagttc ttgccatcct gctacaccaa gttcttg 47

<210> 23

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ccatccgcta caccaagttc ttggctacac caagttcttg 40

<210> 24

<211> 185

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Met Ser Gly Ser Lys Ala Asp Ala Ala Ile Ala Pro Ile Val Met Met

1 5 10 15

Glu His His His Leu Leu Met Asn Trp Asn Lys Pro Ile Asp Leu Ile

20 25 30

Thr Glu Glu Asn Ser Phe Asn His Asn Pro His Phe Ile Val Asp Pro

35 40 45

Pro Ser Glu Thr Leu Ser His Phe Gln Pro Pro Pro Thr Ile Phe Ser

50 55 60

Asp His Gly Gly Gly Glu Glu Ala Glu Glu Glu Glu Glu Glu Glu Gly

65 70 75 80

Glu Glu Glu Met Asp Pro Met Lys Lys Met Gln Tyr Ala Ile Ala Ala

85 90 95

Met Gln Pro Val Asp Leu Asp Pro Ala Thr Val Pro Lys Pro Asn Arg

100 105 110

Arg Asn Val Arg Val Ser Asp Asp Pro Gln Thr Val Val Ala Arg Arg

115 120 125

Arg Arg Glu Arg Ile Ser Glu Lys Ile Arg Ile Leu Lys Arg Met Val

130 135 140

Pro Gly Gly Ala Lys Met Asp Thr Ala Ser Met Leu Asp Glu Ala Ile

145 150 155 160

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

165 170 175

Ser His Ser Ala Trp Ser Ser Tyr Val

180 185

<210> 25

<211> 195

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Met Ser Gly Ser Lys Ala Asp Ala Ala Ala Ile Ala Pro Ile Val Met

1 5 10 15

Met Glu Pro His His Leu Leu Met Asn Trp Asn Lys Pro Ile Asp Leu

20 25 30

Ile Thr Gln Glu Asn Ser Phe Asn His Asn Pro His Phe Met Val Asp

35 40 45

Pro Pro Ser Glu Thr Leu Ser His Phe Gln Pro Pro Pro Thr Val Phe

50 55 60

Ser Asp His Gly Gly Gly Glu Glu Ala Glu Asp Glu Glu Gly Glu Glu

65 70 75 80

Glu Met Asp Glu Met Lys Glu Met Gln Tyr Ala Ile Ala Ala Met Gln

85 90 95

Pro Val Asp Ile Asp Pro Ala Thr Val Pro Lys Pro Asn Arg Arg Asn

100 105 110

Val Arg Val Ser Glu Asp Pro Gln Thr Val Val Ala Arg Arg Arg Arg

115 120 125

Glu Arg Ile Ser Glu Lys Ile Arg Ile Leu Lys Arg Met Val Pro Gly

130 135 140

Gly Ala Lys Met Asp Thr Ala Ser Met Leu Asp Glu Ala Ile Arg Tyr

145 150 155 160

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

165 170 175

Leu Gly Ala Pro Met Ser Asp Pro Ser Cys Leu Cys Tyr Tyr His Asn

180 185 190

Ser Asp Thr

195

<210> 26

<211> 2256

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gggtgaggta tctccatttc aattcttctc tttatatatt aatcgaatta tttacgtatg 60

aaatgaacgt ttatatagaa atttcgtgtg gaaaacgaca tgtacacggc atctcaagac 120

caattagtaa tatactttag tggtgattac atgtttactt atccaattga gaatttaaag 180

catcgacaat accttaatgt cgattaagcc gtccccactt catgtaatga gttatggggg 240

gagagagaga tcccgaaatt cgtcaaataa aacaacttag aactaaaaac cgacaccaag 300

tatcataaag gaaatgttga agaagtcatt tatcgtatcc agctcacaat tcctaagatt 360

aaatcatgac cgttggaaga gcttataaga ttaaactgaa gaaattgtgg gttttagaag 420

aaagacaaga aagagaagaa catgatctta cattgcctat tttggtgtat aggagttgtc 480

aaaaagagga gagagaggag acaattaggt caaataaatg agcactaaaa atggagacat 540

gtgttgagta actattacaa gagcgactta tgcttctata tggcaatgat atcatcacca 600

aagtgcaatg cccctttttg ccctagtttc gtaaagtctc tctccttctt cgtccttagg 660

aaaaacccta aattaaatcc tgtgttcttg atctttcttt ttgagtaacc atgattttga 720

ccacacacta gttcttctat attttgtggt ctataggatt ttgctttata tgtgtttctt 780

gtattgctcc gtacgtgcgt atataaattt aaatggttac aacaaggttt attataaata 840

ggcacaaatt agtccatgaa gttatttagc ttgcacaagt ataatttgtt aagtatttaa 900

atatataaat ttgttacaaa acttaattaa atttatctga ttatattttc tttagtgttc 960

ttcctttgcc aacgttgagg tagctattat tattattatt ttgaacatta tgtacgtagt 1020

tatcttggct agttatgatt cgaattctta atttggatca cacttaacag tatttaaaat 1080

attcttagaa ctaaaataat taagagttac ctttaaattg aagtattcgt gctaaacaga 1140

aactagaata aacaaatgat tgcatgttaa tttttttttt cgattttcct atcagaataa 1200

acacatgatt gcatgcaaat tttgtttttg attacgttat cttttgttta ttttagtttt 1260

gatgctaatt aatatttttt attaacaact cacatacatt ctacctgatt ctaggtcaga 1320

taatgacaca gcgcaacaaa attaatacaa aaccttcgga aagtagaata ccgcagaagt 1380

aacttttttg ggtacatacg aaatacagtg aaatctctat aaattaataa tgttgggact 1440

ataccaaaac tataattttt tattaattta tagagattaa tttatcgata tactaattga 1500

atcaaaaact taatttgaga ctaaaaaatt atattatttt atagagattt ttagtgtata 1560

ttaatttata gaatattatt ttataaaaaa ttttagtgtg tattaattta tagagtatta 1620

atttaaagag gttatactgt aatgtgaatc ttcgaaaaac atgccataca taaccacgga 1680

tcatagtcga cacctcaacg tgaagcaaat ttgacaattt acatacataa ccaacaaaaa 1740

gtagaatacc ttgaaaattt aaaacccaaa atatgatgta aaactcaagc ttggtccaga 1800

gcataaaaaa attaaagcca tcgctttggt atcacatatt taaacgtcag tttttttttt 1860

tttttttttt gggggggggg ggggggtaat ataaaaatat aattaacaaa aaaaaattat 1920

gaaacaatta gcatgtaaaa cactaatctt ttggttgtga caaaacgttt tcacaaatgt 1980

tctataaata aattcaagtg cattttatct gcaaaatata tactttcact cataaaataa 2040

gagcgtttaa aacattcata cacgcactac attgacatga caaaagaaat ccgcaaatac 2100

acatgatgta tgtcgaaaaa aacaaaaaat acacatgatg tatatataga gaggatagta 2160

tctaggaaat aagactatat tatatatata aagaaaatag agaaaagata aaaatataaa 2220

ttggtatgta taaaagaaag gtctatgcgt ctctag 2256

<210> 27

<211> 1242

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ctagagacgc atagaccttt cttttataca taccaaaatt tttttctcta atttctttat 60

atatataata tagtcttatt tcctagatat atccgaacta aatatgtttg tatttgcgga 120

tttcttttgt catgtcaatc tagtgcgtat atgaatgttt taaacgctct tattttatga 180

gtgaaagtat atattttgca gataaaatgt gcttgaattt atttatagaa catttgtgaa 240

aacgttttgt cacaaccaaa agactagtgt tttacatgct aattatttca taaattttct 300

tgatatttat atttttatat tacttcccca aaaaaaaaaa ctgacgttta aatatctgat 360

accaaagcga tggctttaat ttttttatgc tctggaccaa gcttgagttt tacatcatat 420

tttgggttta ggttttcacg gtattctact ttttgttggt tatgtatgta gattgtcaaa 480

tttgcttcac gttgaggtgt cgactatgat ccgtggttat gtcgtatggc atgatttttg 540

aagattcaaa ctacttcgta tgtctaccca aaaatgttac ttccgcggta atctactttc 600

cgaaagtttt gtataatttt gttgcgctgt gtcattatct gacctagaat caggtagaat 660

gtatggaagt tgttaataaa aaaatattaa ttagcatcaa aactaaaata aacaaaagat 720

aacataatca aaaacaaaat ttgcatgcaa tcatgtgttt attctgatat ataggatatt 780

cgaaaaaaaa aataacatgc aatcatttgt ttattgtagt ttctgtttaa cacgaatact 840

tcaatttcaa gttaactctt aattatttta gtactaagaa tattttaaat agtatttttt 900

taaatactgt taagtgttat ccaaattaag aatttgaatc ataactagcc aaaataacta 960

cgtacataat acataatgtt caaaataata ataataataa taataataat aataataata 1020

gctacctcaa ctttggcaaa tgaagaacac taaagaaaat ataatcagat aaagttaatc 1080

aagttttgta gcaaatttat atatttaaat acttaacaca cacacacaca cacacattta 1140

tatacctctt gtgcaagcta aataacctca tggactaatt tgtgcctgtt tataataaac 1200

cttaattgtt gtaaccattt aaatttatat acgcacgtac gg 1242

<210> 28

<211> 550

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

atgtctggct caaaagcaga tgcagccata gccccaatag tcatgatgga gcatcatcat 60

ctccttatga attggaacaa acctattgat ctcattacag aagaaaactc ttttaaccac 120

aatcctcatt tcatagtaga tccaccttcc gaaaccctaa gccacttcca gcccccgccg 180

acaatcttct ccgatcacgg aggaggagag gaagcagaag aagaagaaga agaagaagga 240

gaggaagaga tggatccgat gaagaagatg caatacgcga ttgctgccat gcagcccgta 300

gacctcgatc cagccaccgt tcctaagccg aaccgccgta acgtaagggt aagcgacgac 360

cctcagacgg tggtggctcg tcggcgtaga gaaaggataa gcgagaagat ccggatattg 420

aagaggatgg tgccaggcgg tgcaaagatg gacactgcct ccatgctcga cgatggacac 480

caagttcttg aaacggcagg tgaggctagc ttcttcagcc tcacactcag cttggagctc 540

ctatgtctga 550

<210> 29

<211> 559

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

atgtctggct caaaagcaga tgcagccata gccccaatag tcatgatgga gcatcatcat 60

ctccttatga attggaacaa acctattgat ctcattacag aagaaaactc ttttaaccac 120

aatcctcatt tcatagtaga tccaccttcc gaaaccctaa gccacttcca gcccccgccg 180

acaatcttct ccgatcacgg aggaggagag gaagcagaag aagaagaaga agaagaagga 240

gaggaagaga tggatccgat gaagaagatg caatacgcga ttgctgccat gcagcccgta 300

gacctcgatc cagccaccgt tcctaagccg aaccgccgta acgtaagggt aagcgacgac 360

cctcagacgg tggtggctcg tcggcgtaga gaaaggataa gcgagaagat ccggatattg 420

aagaggatgg tgccaggcgg tgcaaagatg gacactgcct ccatgctcga cgaagccatc 480

cagctacacc aagttcttga aacggcaggt gaggctagct tcttcagcct cacactcagc 540

ttggagctcc tatgtctga 559

<210> 30

<211> 589

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

atgtctggtt caaaagcaga tgcagcagcc atagctccaa tagtcatgat ggagcctcat 60

catctcctta tgaactggaa caaacctatt gatctcatta cacaagaaaa ctcttttaac 120

cacaatcctc atttcatggt agatccacct tccgaaaccc taagccactt ccagcccccg 180

ccgacagtct tctccgatca cggaggagga gaggaagcag aagacgaaga aggagaggaa 240

gagatggatg agatgaagga gatgcaatac gcgattgctg ccatgcagcc cgtagacatc 300

gatccagcca ccgttcctaa gccgaaccgc cgtaacgtaa gggtaagcga ggacccccag 360

acggtggtgg ctcgtcggcg tagagaaagg ataagcgaga agatccggat attgaagagg 420

atggtgccag gcggtgcaaa gatggacact gcctccatgc tcgacgaagc catccagcta 480

caccaagttc ttgaaacggc aggtgaggct tcttcagcct cacactcagc ttggggctcc 540

tatgtctgac ccttcttgcc tttgttatta ccacaactcg gatacctaa 589

Claims

1. Gene for preventing cabbage type rape from cracking hornBnINDDouble mutant obtained by mutation, characterized in that said double mutant consists ofBnA03.INDAndBnC03.INDsimultaneously carrying out nucleotide sequence mutation in a gene coding region, wherein the plant containing the mutation has a crack resistance shape;

BnA03.INDthe nucleotide sequence of (A) is SEQ ID NO. 12,BnC03.INDhas the nucleotide sequence of SEQ ID NO. 13,BnA03.INDandBnC03.INDthe nucleotide sequences of the double mutants which generate the anti-cracking angle phenotype after mutation are respectively SEQ ID NO. 29 and SEQ ID NO. 30.

2. Gene for preventing cabbage type rape from cracking hornBnINDSingle mutant obtained by mutation, characterized in that said single mutant consists ofBnA03.INDThe nucleotide sequence mutation in the gene coding region occurs, the plant containing the mutated nucleotide sequence has the shape of crack resistance angle,BnA03.INDthe nucleotide sequence of (A) is SEQ ID NO. 12,BnA03.INDthe nucleotide sequence of the single mutant which generates the anti-cracking angle phenotype after mutation is SEQ ID NO. 28.

3. Use of the double mutant of claim 1 or the single mutant of claim 2 for dehiscence-resistant improved breeding of brassica napus.