CN109879945A - The function and application of cabbage type rape cracking resistance angle gene BnIND - Google Patents

Abstract

The invention belongs to rape molecular breeding technical fields, disclose the function and application of a cabbage type rape cracking resistance angle gene BnIND, there are the functions of partial redundance in siliqua of oilseed rape dehiscence process for two homologous copies of the gene, and BnA03.IND copy is bigger compared with the effect played in silique dehiscence process of BnC03.IND copy；Sequence comparing analysis finds that two copies of BnIND gene encode albumen with the same function, and promoter region has biggish sequence variations；Since the sequence difference of promoter region causes two of BnIND gene to copy the difference occurred on gene expression amount, BnA03.IND copy is eventually led to compared with BnC03.IND copy and plays bigger effect in siliqua of oilseed rape dehiscence process.The present invention provides important functional gene and breeding material for the genetic improvement of rape cracking resistance angle character.

Description

The function and application of cabbage type rape cracking resistance angle gene BnIND

Technical field

The invention belongs to rape molecular breeding fields, are related to a cabbage type rape gene BnIND in terms of silique cracking resistance Function and application.

Background technique

The cracking of mature silique is the one kind of rape during long-term evolution in order to adapt to environment, produce offspring and formed Biological characteristics.But the easy to cracking of silique is unfavorable character in Rape-seed production, will lead to the rape underproduction, is also unfavorable for machine The popularization and application of toolization harvest.According to statistics, annual resulting production loss typically constitutes from about the 20% of total output, in severe day Up to 50% under the conditions of gas (Price etc., 1996；Child etc., 1998).Therefore, the cracking resistance angle oil for being suitble to mechanized harvest is developed Vegetable kind will bring significant economic benefit.

In Cruciferae (Brassicaceae) plant including arabidopsis and rape, the cracking of silique is by one A little specialized cells are developed between two fruit lobes caused by absciss layer area.Absciss layer area is made of lignifying floor and separating layer；In silique When mature, lignifying layer is hardened, the hydrolase of separating layer secretion simultaneously leads to the reduction of its breaking resistance, their collective effect causes Silique cracks from absciss layer.

In arabidopsis, gene relevant to silique cracking and its regulated and control network also have a large amount of research.Wherein, two function Can redundancy MADS-box gene SHATTERPROOF1 (SHP1) and SHP2 participation fruit lobe edge development (Liljegren etc., 2000)；They regulate and control the expression of downstream INDEHISCENT (IND) gene.The afunction mutant of these genes is all unable to shape At normal fruit lobe edge, cause silique that cannot crack, seed can not shed.The Rape pod development and arabidopsis ten of cabbage type rape Seemingly, therefore the cracking resistance Mechanism Study in arabidopsis provides information abundant for the correlative study in rape to split-phase.

Cabbage type rape is a kind of allotetraploid species, is by two diploid progenitors kind Chinese cabbage (B.rapa, AA genes Group) and wild cabbage (B.oleracea, CC genome) natural hybrization after evolve (Chalhoub etc., 2014).Cabbage type rape Silique be easy cracking, traditional method mainly by interspecific hybridization is introduced into the Crack resistant base in sibling species because raising resistance (Prakash and Chopra 1988；Morgan etc., 1998；Summers etc., 2003).

In conclusion problem of the existing technology is:

The method of the prior art takes a long time, and can also introduce some other unfavorable economical character simultaneously, so that they are not It is suitble to growth condition (Summers etc., 2003).Compared with interspecific hybridization method, will can fast and effeciently it be intended by genetic transformation Crack resistant base in southern mustard has successfully applied to the something lost of silique cracking resistance in Brassica Crops because importeding into excellent kind Biography improvement (Chandler etc., 2005；Deng 2006), but this method at present many countries and regions by turn The limitation of gene policy.Other than interspecific hybridization and transgenosis, it is anti-to improve new hereditary variation can also to be generated by mutagenesis Split angle property.For example, Braatz etc. (2018) is by the mutagenic obtained cracking resistance cornicult variant of EMS, and confirm only to copy ind is bis- Cracking resistance angle property increases in mutant, shows that two copies of the gene have function conservative and the feature of redundancy.However, luring at random Change also results in a large amount of background mutation and has an adverse effect to other characters.Such as the list of two kinds of random mutagenesis of ind is prominent The all extremely significant reduction (Braatz etc., 2018) of variant fertility.In cabbage type rape carry out random mutagenesis another mainly lack The characteristic being trapped in its polyploid causes most of genes to have multiple intimate homologous copies, and random mutagenesis is usually only One of energy random mutation, therefore generally will not all generate apparent phenotype.

Solve the meaning of above-mentioned technical problem:

In recent years, CRISPR/Cas9 successfully applies to as a kind of efficient fixed point target gene editing technique In rape.The present invention targets BnIND homologous gene using CRISPR/Cas9 technology, obtains mutant single plant by genetic transformation, It is separated by inbreeding of more generation, finally obtains the homozygous mutant of the homozygous and double copy of single copy, and to these mutation type surfaces Identification, genetic analysis and the measurement of cracking resistance angle property.Result of study shows double copy mutant of BnIND gene and single The Mutants homozygous of BnA03.IND copy mutation can produce the silique that cracking resistance angle property dramatically increases.BnIND gene is for rape The improvement of silique cracking form has huge application potential and prospect, and new genetic resources are provided for yield of rape breeding.

Summary of the invention

In view of the problems of the existing technology, the present invention provides a kind of cabbage type rape gene BnIND in silique cracking resistance The function and application of aspect, while it is same to provide a kind of two using gene editing technical appraisement cabbage type rape BnIND gene Source copies the function in terms of silique cracking resistance.The present invention targets two homologous copies of cabbage type rape BnIND gene, quickly high Effect obtains the apparent cabbage type rape germ plasm resource of cracking resistance angle phenotype that can stablize heredity, to cracking resistance angle cabbage type rape Breeding has great importance.The mutant is compared without containing T-DNA insertion with wild type, and cracking resistance angle index has reached extremely aobvious It writes horizontal.

The invention is realized in this way a kind of cabbage type rape cracking resistance angle gene BnIND, cabbage type rape cracking resistance angle Candidate gene BnIND nucleotides sequence is classified as SEQ ID NO:12, SEQ ID NO:13.

Another object of the present invention is to provide a kind of cabbage type rape cracking resistance angle gene BnIND coding albumen, The amino acid sequence of the albumen are as follows: SEQ ID NO:24, SEQ ID NO:25.

Another object of the present invention is to provide a kind of control for separating cabbage type rape cracking resistance angle gene BnIND is sweet The promoter at blue type rape cracking resistance angle, the nucleotides sequence of the sequence promoter are classified as SEQ ID NO:26, SEQ ID NO:27.

It is mutated another object of the present invention is to provide a kind of using cabbage type rape cracking resistance angle gene BnIND Double-mutant, the double-mutant is by two of BnIND copies while the nucleotide sequence mutation in gene coding region occurs, Plant containing the mutation has cracking resistance angle character, and the double-mutant nucleotide sequence of cracking resistance angle phenotype is generated 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.

It is mutated another object of the present invention is to provide a kind of using cabbage type rape cracking resistance angle gene BnIND Single mutation, the single mutation occurs the nucleotide sequence mutation in gene coding region by the single copy of BnIND, contains mutation The plant of nucleotide sequence has cracking resistance angle character afterwards, and the single mutation nucleotides sequence that cracking resistance angle phenotype is generated after mutation is classified as SEQ ID NO:28.

Another object of the present invention is to provide a kind of applications using double mutation in breeding.

Another object of the present invention is to provide kind to utilize application of the single mutation in breeding.

Another object of the present invention is to provide a kind of using cabbage type rape cracking resistance angle gene BnIND in Wild cabbage type The application of rape cracking resistance angle character improvement kind.

In the present invention, cabbage type rape cracking resistance angle candidate gene BnIND, nucleotides sequence list such as sequence are as follows:

>BnA03.IND

ATGTCTGGCTCAAAAGCAGATGCAGCCATAGCCCCAATAGTCATGATGGAGCATCATCATCTCCTTAT GAATTGGAACAAACCTATTGATCTCATTACAGAAGAAAACTCTTTTAACCACAATCCTCATTTCATAGTAGATCCA CCTTCCGAAACCCTAAGCCACTTCCAGCCCCCGCCGACAATCTTCTCCGATCACGGAGGAGGAGAGGAAGCAGAAG AAGAAGAAGAAGAAGAAGGAGAGGAAGAGATGGATCCGATGAAGAAGATGCAATACGCGATTGCTGCCATGCAGCC CGTAGACCTCGATCCAGCCACCGTTCCTAAGCCGAACCGCCGTAACGTAAGGGTAAGCGACGACCCTCAGACGGTG GTGGCTCGTCGGCGTAGAGAAAGGATAAGCGAGAAGATCCGGATATTGAAGAGGATGGTGCCAGGCGGTGCAAAGA TGGACACTGCCTCCATGCTCGACGAAGCCATCCGCTACACCAAGTTCTTGAAACGGCAGGTGAGGCTAGCTTCTTC AGCCTCACACTCAGCTTGGAGCTCCTATGTCTGA SEQ ID NO:12.

>BnC03.IND

ATGTCTGGTTCAAAAGCAGATGCAGCAGCCATAGCTCCAATAGTCATGATGGAGCCTCATCATCTCCT TATGAACTGGAACAAACCTATTGATCTCATTACACAAGAAAACTCTTTTAACCACAATCCTCATTTCATGGTAGAT CCACCTTCCGAAACCCTAAGCCACTTCCAGCCCCCGCCGACAGTCTTCTCCGATCACGGAGGAGGAGAGGAAGCAG AAGACGAAGAAGGAGAGGAAGAGATGGATGAGATGAAGGAGATGCAATACGCGATTGCTGCCATGCAGCCCGTAGA CATCGATCCAGCCACCGTTCCTAAGCCGAACCGCCGTAACGTAAGGGTAAGCGAGGACCCCCAGACGGTGGTGGCT CGTCGGCGTAGAGAAAGGATAAGCGAGAAGATCCGGATATTGAAGAGGATGGTGCCAGGCGGTGCAAAGATGGACA CTGCCTCCATGCTCGACGAAGCCATCCGCTACACCAAGTTCTTGAAACGGCAGGTGAGGCTTCTTCAGCCTCACAC TCAGCTTGGGGCTCCTATGTCTGACCCTTCTTGCCTTTGTTATTACCACAACTCGG ATACCTAA SEQ ID NO: 13.

The amino acid sequence of albumen is such as

>BnA03.IND

MSGSKADAAIAPIVMMEHHHLLMNWNKPIDLITEENSFNHNPHFIVDPPSETLSHFQPPPTIFSDHGG GEEAEEEEEEEGEEEMDPMKKMQYAIAAMQPVDLDPATVPKPNRRNVRVSDDPQTVVARRRRERISEKIRILKRMV PGGAKMDTASMLDEAIRYTKFLKRQVRLASSASHSAWSSYV SEQ ID NO:24.

>BnC03.IND

MSGSKADAAAIAPIVMMEPHHLLMNWNKPIDLITQENSFNHNPHFMVDPPSETLSHFQPPPTVFSDHG GGEEAEDEEGEEEMDEMKEMQYAIAAMQPVDIDPATVPKPNRRNVRVSEDPQTVVARRRRERISEKIRILKRMVPG GAKMDTASMLDEAIRYTKFLKRQVRLLQPHTQLGAPMSDPSCLCYYHNSDT SEQ ID NO:25.

Promoter sequence are as follows:

>BnA03.IND

GGGTGAGGTATCTCCATTTCAATTCTTCTCTTTATATATTAATCGAATTATTTACGTATGAAATGAAC GTTTATATAGAAATTTCGTGTGGAAAACGACATGTACACGGCATCTCAAGACCAATTAGTAATATACTTTAGTGGT GATTACATGTTTACTTATCCAATTGAGAATTTAAAGCATCGACAATACCTTAATGTCGATTAAGCCGTCCCCACTT CATGTAATGAGTTATGGGGGGAGAGAGAGATCCCGAAATTCGTCAAATAAAACAACTTAGAACTAAAAACCGACAC CAAGTATCATAAAGGAAATGTTGAAGAAGTCATTTATCGTATCCAGCTCACAATTCCTAAGATTAAATCATGACCG TTGGAAGAGCTTATAAGATTAAACTGAAGAAATTGTGGGTTTTAGAAGAAAGACAAGAAAGAGAAGAACATGATCT TACATTGCCTATTTTGGTGTATAGGAGTTGTCAAAAAGAGGAGAGAGAGGAGACAATTAGGTCAAATAAATGAGCA CTAAAAATGGAGACATGTGTTGAGTAACTATTACAAGAGCGACTTATGCTTCTATATGGCAATGATATCATCACCA AAGTGCAATGCCCCTTTTTGCCCTAGTTTCGTAAAGTCTCTCTCCTTCTTCGTCCTTAGGAAAAACCCTAAATTAA ATCCTGTGTTCTTGATCTTTCTTTTTGAGTAACCATGATTTTGACCACACACTAGTTCTTCTATATTTTGTGGTCT ATAGGATTTTGCTTTATATGTGTTTCTTGTATTGCTCCGTACGTGCGTATATAAATTTAAATGGTTACAACAAGGT TTATTATAAATAGGCACAAATTAGTCCATGAAGTTATTTAGCTTGCACAAGTATAATTTGTTAAGTATTTAAATAT ATAAATTTGTTACAAAACTTAATTAAATTTATCTGATTATATTTTCTTTAGTGTTCTTCCTTTGCCAACGTTGAGG TAGCTATTATTATTATTATTTTGAACATTATGTACGTAGTTATCTTGGCTAGTTATGATTCGAATTCTTAATTTGG ATCACACTTAACAGTATTTAAAATATTCTTAGAACTAAAATAATTAAGAGTTACCTTTAAATTGAAGTATTCGTGC TAAACAGAAACTAGAATAAACAAATGATTGCATGTTAATTTTTTTTTTCGATTTTCCTATCAGAATAAACACATGA TTGCATGCAAATTTTGTTTTTGATTACGTTATCTTTTGTTTATTTTAGTTTTGATGCTAATTAATATTTTTTATTA ACAACTCACATACATTCTACCTGATTCTAGGTCAGATAATGACACAGCGCAACAAAATTAATACAAAACCTTCGGA AAGTAGAATACCGCAGAAGTAACTTTTTTGGGTACATACGAAATACAGTGAAATCTCTATAAATTAATAATGTTGG GACTATACCAAAACTATAATTTTTTATTAATTTATAGAGATTAATTTATCGATATACTAATTGAATCAAAAACTTA ATTTGAGACTAAAAAATTATATTATTTTATAGAGATTTTTAGTGTATATTAATTTATAGAATATTATTTTATAAAA AATTTTAGTGTGTATTAATTTATAGAGTATTAATTTAAAGAGGTTATACTGTAATGTGAATCTTCGAAAAACATGC CATACATAACCACGGATCATAGTCGACACCTCAACGTGAAGCAAATTTGACAATTTACATACATAACCAACAAAAA GTAGAATACCTTGAAAATTTAAAACCCAAAATATGATGTAAAACTCAAGCTTGGTCCAGAGCATAAAAAAATTAAA GCCATCGCTTTGGTATCACATATTTAAACGTCAGTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGGGGGGGTAATAT AAAAATATAATTAACAAAAAAAAATTATGAAACAATTAGCATGTAAAACACTAATCTTTTGGTTGTGACAAAACGT TTTCACAAATGTTCTATAAATAAATTCAAGTGCATTTTATCTGCAAAATATATACTTTCACTCATAAAATAAGAGC GTTTAAAACATTCATACACGCACTACATTGACATGACAAAAGAAATCCGCAAATACACATGATGTATGTCGAAAAA AACAAAAAATACACATGATGTATATATAGAGAGGATAGTATCTAGGAAATAAGACTATATTATATATATAAAGAAA ATAGAGAAAAGATAAAAATATAAATTGGTATGTATAAAAGAAAGGTCTATGCGTCT CTAG SEQ ID NO:26.

>BnC03.IND

CTAGAGACGCATAGACCTTTCTTTTATACATACCAAAATTTTTTTCTCTAATTTCTTTATATATATAA TATAGTCTTATTTCCTAGATATATCCGAACTAAATATGTTTGTATTTGCGGATTTCTTTTGTCATGTCAATCTAGT GCGTATATGAATGTTTTAAACGCTCTTATTTTATGAGTGAAAGTATATATTTTGCAGATAAAATGTGCTTGAATTT ATTTATAGAACATTTGTGAAAACGTTTTGTCACAACCAAAAGACTAGTGTTTTACATGCTAATTATTTCATAAATT TTCTTGATATTTATATTTTTATATTACTTCCCCAAAAAAAAAAACTGACGTTTAAATATCTGATACCAAAGCGATG GCTTTAATTTTTTTATGCTCTGGACCAAGCTTGAGTTTTACATCATATTTTGGGTTTAGGTTTTCACGGTATTCTA CTTTTTGTTGGTTATGTATGTAGATTGTCAAATTTGCTTCACGTTGAGGTGTCGACTATGATCCGTGGTTATGTCG TATGGCATGATTTTTGAAGATTCAAACTACTTCGTATGTCTACCCAAAAATGTTACTTCCGCGGTAATCTACTTTC CGAAAGTTTTGTATAATTTTGTTGCGCTGTGTCATTATCTGACCTAGAATCAGGTAGAATGTATGGAAGTTGTTAA TAAAAAAATATTAATTAGCATCAAAACTAAAATAAACAAAAGATAACATAATCAAAAACAAAATTTGCATGCAATC ATGTGTTTATTCTGATATATAGGATATTCGAAAAAAAAAATAACATGCAATCATTTGTTTATTGTAGTTTCTGTTT AACACGAATACTTCAATTTCAAGTTAACTCTTAATTATTTTAGTACTAAGAATATTTTAAATAGTATTTTTTTAAA TACTGTTAAGTGTTATCCAAATTAAGAATTTGAATCATAACTAGCCAAAATAACTACGTACATAATACATAATGTT CAAAATAATAATAATAATAATAATAATAATAATAATAATAGCTACCTCAACTTTGGCAAATGAAGAACACTAAAGA AAATATAATCAGATAAAGTTAATCAAGTTTTGTAGCAAATTTATATATTTAAATACTTAACACACACACACACACA CACATTTATATACCTCTTGTGCAAGCTAAATAACCTCATGGACTAATTTGTGCCTGTTTATAATAAACCTTAATTG TTGTAACCATTTAAATTTATATACGCACGTACGG SEQ ID NO:27.

The nucleotide sequence of double-mutant is such as

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 mutation nucleotide sequence is such as

IND-201-4-10 aaCC S4

CCATCCGCT---ACACCAAGTTCTTG wt

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

In the present invention, by cloning the genome sequence of cabbage type rape cracking resistance angle gene BnIND, CRISPR/ is utilized CAS9 technology targets two homologous copies of BnIND, obtains mutant, obtained mutant single plant are 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 present invention carries out cracking resistance angle assessment of indices by the silique to obtained mutant and wild type single plant, finds BnIND The extremely significant increase of cracking resistance angle index of the homozygous mutation of double copy Mutants homozygous of gene and single BnA03.IND copy.

The present invention carries out phenotype and displaing microstructure observing by mutant to acquisition and wild type silique, discovery with it is wild Type is compared, and structure of the Mutants homozygous of double Mutants homozygous and single BnA03.IND copy mutation at fruit lobe edge has not Same institutional framework difference.Sequence comparing analysis finds that two copies of BnIND gene encode albumen with the same function, and Promoter region has biggish sequence variations；Further gene expression analysis confirms BnA03.IND during development The expression quantity of copy is significantly higher than BnC03.IND copy.Thus, it could be seen that the sequence difference due to promoter region leads to BnIND There is the difference on gene expression amount in two copies of gene, eventually lead to BnA03.IND copy and exist compared with BnC03.IND copy Bigger effect is played in siliqua of oilseed rape dehiscence process.

In conclusion advantages of the present invention and good effect are as follows:

The CRISPR/Cas9 that the present invention uses is a kind of efficient fixed point target gene editing technique, the technology specific aim By force, gene knockout is carried out to rape with it, it can acquisition mutant rapidly and efficiently.Mutant is planted, by inbreeding of more generation Separation can obtain the Mutants homozygous without T-DNA insertion.This method more rapidly than traditional crossbreeding technology, than Mutation breeding is safer.

Microexamination discovery is carried out to the mutant silique of acquisition, in interior peel edge, the mutant majority of BnIND Pericarp membrane has the cell wall thicker than wild type.It there's almost no the lignifying layer of peel edge in double-mutant and divide B layers of the endocarp of absciss layer, lignifying is directly connected with the lignifying vascular bundle of diaphragm, and continuous wood is formd around pericarp Matter group prevents the cracking of silique.Although the lignifying structure of single mutant is similar with wild type, there is also some small Difference.It is compared with wild type, single mutant shows one layer small of the non-wood keratinocyte cells to form separating layer.In addition to this, In the mono- Mutants homozygous of BnA03.IND, pericarp and diaphragm are closely joined together by lignifying bridge and prevent opening for silique It splits.But in wild type and BnC03.IND single mutant, in the endocarp layer of lignifying and the diaphragm vascular tissue of lignifying Between exist have apparent separating layer.

This experiment obtains the Brassica napus Mutant Cr germ plasm resource with cracking resistance corner characteristics, compared to wild type, dashes forward The extremely significant increase of variant cracking resistance angle index.The material provides valuable resource for rapeseed breeding plan.

Gene of the invention is the important gene that silique cracking is controlled in cabbage type rape, its two homologous copies are in oil There are the functions of partial redundance in dish silique dehiscence process, and BnA03.IND copy is copied compared with BnC03.IND and cracked in silique Effect played in journey is bigger；Sequence comparing analysis finds that two copies of BnIND gene encode albumen with the same function, And promoter region has biggish sequence variations；Further gene expression analysis confirms during Rape pod development The expression quantity of BnA03.IND copy is significantly higher than BnC03.IND copy；Since the sequence difference of promoter region leads to BnIND There is the difference on gene expression amount in two copies of gene, eventually lead to BnA03.IND copy and exist compared with BnC03.IND copy Bigger effect is played in siliqua of oilseed rape dehiscence process.The present invention provides important function for the genetic improvement of rape cracking resistance angle character It can gene and breeding material.

Detailed description of the invention

Fig. 1 is that utilization CRISPR/CAS9 technology provided in an embodiment of the present invention obtains cracking resistance angle Brassica napus Mutant Cr Method flow diagram.

Fig. 2 is the gene structure figure and carrier figure of BnIND two copies provided in an embodiment of the present invention.

In figure: (1) it includes an exon that white box, which represents the gene, and gray area indicates the bHLH structure of the gene Domain, the vertical line in genetic model indicate that target site, arrow indicate the direction of sgRNA.S1-S4 illustrates target sequence, leukorrhagia The area PAM of scribing line.(2) structure figures of SBnIND carrier.

Fig. 3 is the gene expression figure provided in an embodiment of the present invention in J9707 in different tissues.

Fig. 4 is the measurement result of cracking resistance ascent provided in an embodiment of the present invention, shows the mono- Mutants homozygous of BnA03.IND It is extremely significant higher than wild type with the cracking resistance ascent of double Mutants homozygous.

Fig. 5 is the phenotypic map provided in an embodiment of the present invention for indicating wild type and mutant.

Fig. 6 is 18 periods provided in an embodiment of the present invention in Rape pod development, the slice microexamination figure of silique.

A in figure, b, c, d are the structural schematic diagram before phloroglucinol stain；E, f, g, h are paraffin section phloroglucinol stain The silique fruit lobe edge hair of single Mutants homozygous of effect picture afterwards, BnIND Gene Double Mutants homozygous and BnA03.IND copy It educates abnormal.

Specific embodiment

In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to Limit the present invention.

In the prior art, it is not obtained using the homologous gene of CRISPR/Cas9 technology targeting BnIND by genetic transformation It to mutant single plant, is separated by inbreeding of more generation, obtains the homozygous mutant of double copies.Do not dash forward to BnIND function forfeiture The cracking resistance angle mechanism of variant is illustrated, and the reason of two copies are to technical effect caused by the property of cracking resistance angle is not parsed.

To solve the above problems, below with reference to concrete scheme, the present invention is described in detail.

A kind of cabbage type rape cracking resistance angle gene BnIND provided in an embodiment of the present invention, cabbage type rape cracking resistance angle Candidate gene BnIND nucleotides sequence is classified as SEQ ID NO:12, SEQ ID NO:13.

The embodiment of the present invention provides a kind of albumen of cabbage type rape cracking resistance angle gene BnIND coding, the albumen Amino acid sequence are as follows: SEQ ID NO:23, SEQ ID NO:24.

The embodiment of the present invention provides a kind of control cabbage type rape for separating cabbage type rape cracking resistance angle gene BnIND The promoter at cracking resistance angle, the nucleotides sequence of the sequence promoter are classified as SEQ ID NO:25, SEQ ID NO:26.

The embodiment of the present invention provides a kind of double mutation being mutated using cabbage type rape cracking resistance angle gene BnIND Body, the double-mutant copies while occurring the nucleotide sequence mutation in gene coding region by two of BnIND, containing described The plant of mutation has cracking resistance angle character, the double-mutant nucleotide sequence of cracking resistance angle phenotype is generated 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.

It is prominent that the embodiment of the present invention provides a kind of list being mutated using cabbage type rape cracking resistance angle gene BnIND Become, by the single copy of BnIND the nucleotide sequence mutation in gene coding region occurs for the single mutation, contains nucleosides after mutation The plant of acid sequence has cracking resistance angle character, and the single mutation nucleotides sequence that cracking resistance angle phenotype is generated after mutation is classified as SEQ ID NO: 28。

In the present invention, cabbage type rape cracking resistance angle candidate gene BnIND, nucleotides sequence list such as sequence are as follows:

>BnA03.IND

ATGTCTGGCTCAAAAGCAGATGCAGCCATAGCCCCAATAGTCATGATGGAGCATCATCATCTCCTTAT GAATTGGAACAAACCTATTGATCTCATTACAGAAGAAAACTCTTTTAACCACAATCCTCATTTCATAGTAGATCCA CCTTCCGAAACCCTAAGCCACTTCCAGCCCCCGCCGACAATCTTCTCCGATCACGGAGGAGGAGAGGAAGCAGAAG AAGAAGAAGAAGAAGAAGGAGAGGAAGAGATGGATCCGATGAAGAAGATGCAATACGCGATTGCTGCCATGCAGCC CGTAGACCTCGATCCAGCCACCGTTCCTAAGCCGAACCGCCGTAACGTAAGGGTAAGCGACGACCCTCAGACGGTG GTGGCTCGTCGGCGTAGAGAAAGGATAAGCGAGAAGATCCGGATATTGAAGAGGATGGTGCCAGGCGGTGCAAAGA TGGACACTGCCTCCATGCTCGACGAAGCCATCCGCTACACCAAGTTCTTGAAACGGCAGGTGAGGCTAGCTTCTTC AGCCTCACACTCAGCTTGGAGCTCCTATGTCTGA SEQ ID NO:12.

>BnC03.IND

ATGTCTGGTTCAAAAGCAGATGCAGCAGCCATAGCTCCAATAGTCATGATGGAGCCTCATCATCTCCT TATGAACTGGAACAAACCTATTGATCTCATTACACAAGAAAACTCTTTTAACCACAATCCTCATTTCATGGTAGAT CCACCTTCCGAAACCCTAAGCCACTTCCAGCCCCCGCCGACAGTCTTCTCCGATCACGGAGGAGGAGAGGAAGCAG AAGACGAAGAAGGAGAGGAAGAGATGGATGAGATGAAGGAGATGCAATACGCGATTGCTGCCATGCAGCCCGTAGA CATCGATCCAGCCACCGTTCCTAAGCCGAACCGCCGTAACGTAAGGGTAAGCGAGGACCCCCAGACGGTGGTGGCT CGTCGGCGTAGAGAAAGGATAAGCGAGAAGATCCGGATATTGAAGAGGATGGTGCCAGGCGGTGCAAAGATGGACA CTGCCTCCATGCTCGACGAAGCCATCCGCTACACCAAGTTCTTGAAACGGCAGGTGAGGCTTCTTCAGCCTCACAC TCAGCTTGGGGCTCCTATGTCTGACCCTTCTTGCCTTTGTTATTACCACAACTCGG ATACCTAA SEQ ID NO: 13.

The amino acid sequence of albumen is such as

>BnA03.IND

MSGSKADAAIAPIVMMEHHHLLMNWNKPIDLITEENSFNHNPHFIVDPPSETLSHFQPPPTIFSDHGG GEEAEEEEEEEGEEEMDPMKKMQYAIAAMQPVDLDPATVPKPNRRNVRVSDDPQTVVARRRRERISEKIRILKRMV PGGAKMDTASMLDEAIRYTKFLKRQVRLASSASHSAWSSYV SEQ ID NO:24.

>BnC03.IND

MSGSKADAAAIAPIVMMEPHHLLMNWNKPIDLITQENSFNHNPHFMVDPPSETLSHFQPPPTVFSDHG GGEEAEDEEGEEEMDEMKEMQYAIAAMQPVDIDPATVPKPNRRNVRVSEDPQTVVARRRRERISEKIRILKRMVPG GAKMDTASMLDEAIRYTKFLKRQVRLLQPHTQLGAPMSDPSCLCYYHNSDT SEQ ID NO:25.

Promoter sequence are as follows:

>BnA03.IND

GGGTGAGGTATCTCCATTTCAATTCTTCTCTTTATATATTAATCGAATTATTTACGTATGAAATGAAC GTTTATATAGAAATTTCGTGTGGAAAACGACATGTACACGGCATCTCAAGACCAATTAGTAATATACTTTAGTGGT GATTACATGTTTACTTATCCAATTGAGAATTTAAAGCATCGACAATACCTTAATGTCGATTAAGCCGTCCCCACTT CATGTAATGAGTTATGGGGGGAGAGAGAGATCCCGAAATTCGTCAAATAAAACAACTTAGAACTAAAAACCGACAC CAAGTATCATAAAGGAAATGTTGAAGAAGTCATTTATCGTATCCAGCTCACAATTCCTAAGATTAAATCATGACCG TTGGAAGAGCTTATAAGATTAAACTGAAGAAATTGTGGGTTTTAGAAGAAAGACAAGAAAGAGAAGAACATGATCT TACATTGCCTATTTTGGTGTATAGGAGTTGTCAAAAAGAGGAGAGAGAGGAGACAATTAGGTCAAATAAATGAGCA CTAAAAATGGAGACATGTGTTGAGTAACTATTACAAGAGCGACTTATGCTTCTATATGGCAATGATATCATCACCA AAGTGCAATGCCCCTTTTTGCCCTAGTTTCGTAAAGTCTCTCTCCTTCTTCGTCCTTAGGAAAAACCCTAAATTAA ATCCTGTGTTCTTGATCTTTCTTTTTGAGTAACCATGATTTTGACCACACACTAGTTCTTCTATATTTTGTGGTCT ATAGGATTTTGCTTTATATGTGTTTCTTGTATTGCTCCGTACGTGCGTATATAAATTTAAATGGTTACAACAAGGT TTATTATAAATAGGCACAAATTAGTCCATGAAGTTATTTAGCTTGCACAAGTATAATTTGTTAAGTATTTAAATAT ATAAATTTGTTACAAAACTTAATTAAATTTATCTGATTATATTTTCTTTAGTGTTCTTCCTTTGCCAACGTTGAGG TAGCTATTATTATTATTATTTTGAACATTATGTACGTAGTTATCTTGGCTAGTTATGATTCGAATTCTTAATTTGG ATCACACTTAACAGTATTTAAAATATTCTTAGAACTAAAATAATTAAGAGTTACCTTTAAATTGAAGTATTCGTGC TAAACAGAAACTAGAATAAACAAATGATTGCATGTTAATTTTTTTTTTCGATTTTCCTATCAGAATAAACACATGA TTGCATGCAAATTTTGTTTTTGATTACGTTATCTTTTGTTTATTTTAGTTTTGATGCTAATTAATATTTTTTATTA ACAACTCACATACATTCTACCTGATTCTAGGTCAGATAATGACACAGCGCAACAAAATTAATACAAAACCTTCGGA AAGTAGAATACCGCAGAAGTAACTTTTTTGGGTACATACGAAATACAGTGAAATCTCTATAAATTAATAATGTTGG GACTATACCAAAACTATAATTTTTTATTAATTTATAGAGATTAATTTATCGATATACTAATTGAATCAAAAACTTA ATTTGAGACTAAAAAATTATATTATTTTATAGAGATTTTTAGTGTATATTAATTTATAGAATATTATTTTATAAAA AATTTTAGTGTGTATTAATTTATAGAGTATTAATTTAAAGAGGTTATACTGTAATGTGAATCTTCGAAAAACATGC CATACATAACCACGGATCATAGTCGACACCTCAACGTGAAGCAAATTTGACAATTTACATACATAACCAACAAAAA GTAGAATACCTTGAAAATTTAAAACCCAAAATATGATGTAAAACTCAAGCTTGGTCCAGAGCATAAAAAAATTAAA GCCATCGCTTTGGTATCACATATTTAAACGTCAGTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGGGGGGGTAATAT AAAAATATAATTAACAAAAAAAAATTATGAAACAATTAGCATGTAAAACACTAATCTTTTGGTTGTGACAAAACGT TTTCACAAATGTTCTATAAATAAATTCAAGTGCATTTTATCTGCAAAATATATACTTTCACTCATAAAATAAGAGC GTTTAAAACATTCATACACGCACTACATTGACATGACAAAAGAAATCCGCAAATACACATGATGTATGTCGAAAAA AACAAAAAATACACATGATGTATATATAGAGAGGATAGTATCTAGGAAATAAGACTATATTATATATATAAAGAAA ATAGAGAAAAGATAAAAATATAAATTGGTATGTATAAAAGAAAGGTCTATGCGTCT CTAG SEQ ID NO:26.

>BnC03.IND

CTAGAGACGCATAGACCTTTCTTTTATACATACCAAAATTTTTTTCTCTAATTTCTTTATATATATAA TATAGTCTTATTTCCTAGATATATCCGAACTAAATATGTTTGTATTTGCGGATTTCTTTTGTCATGTCAATCTAGT GCGTATATGAATGTTTTAAACGCTCTTATTTTATGAGTGAAAGTATATATTTTGCAGATAAAATGTGCTTGAATTT ATTTATAGAACATTTGTGAAAACGTTTTGTCACAACCAAAAGACTAGTGTTTTACATGCTAATTATTTCATAAATT TTCTTGATATTTATATTTTTATATTACTTCCCCAAAAAAAAAAACTGACGTTTAAATATCTGATACCAAAGCGATG GCTTTAATTTTTTTATGCTCTGGACCAAGCTTGAGTTTTACATCATATTTTGGGTTTAGGTTTTCACGGTATTCTA CTTTTTGTTGGTTATGTATGTAGATTGTCAAATTTGCTTCACGTTGAGGTGTCGACTATGATCCGTGGTTATGTCG TATGGCATGATTTTTGAAGATTCAAACTACTTCGTATGTCTACCCAAAAATGTTACTTCCGCGGTAATCTACTTTC CGAAAGTTTTGTATAATTTTGTTGCGCTGTGTCATTATCTGACCTAGAATCAGGTAGAATGTATGGAAGTTGTTAA TAAAAAAATATTAATTAGCATCAAAACTAAAATAAACAAAAGATAACATAATCAAAAACAAAATTTGCATGCAATC ATGTGTTTATTCTGATATATAGGATATTCGAAAAAAAAAATAACATGCAATCATTTGTTTATTGTAGTTTCTGTTT AACACGAATACTTCAATTTCAAGTTAACTCTTAATTATTTTAGTACTAAGAATATTTTAAATAGTATTTTTTTAAA TACTGTTAAGTGTTATCCAAATTAAGAATTTGAATCATAACTAGCCAAAATAACTACGTACATAATACATAATGTT CAAAATAATAATAATAATAATAATAATAATAATAATAATAGCTACCTCAACTTTGGCAAATGAAGAACACTAAAGA AAATATAATCAGATAAAGTTAATCAAGTTTTGTAGCAAATTTATATATTTAAATACTTAACACACACACACACACA CACATTTATATACCTCTTGTGCAAGCTAAATAACCTCATGGACTAATTTGTGCCTGTTTATAATAAACCTTAATTG TTGTAACCATTTAAATTTATATACGCACGTACGG SEQ ID NO:27.

The nucleotide sequence of double-mutant is such as

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 mutation nucleotide sequence is such as

IND-201-4-10 aaCC S4

CCATCCGCTACACCAAGTTCTTG

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

The invention will be further described combined with specific embodiments below.

Embodiment

It is provided in an embodiment of the present invention to obtain cracking resistance angle Brassica napus Mutant Cr single plant using CRISPR/CAS9 technology, The cracking resistance angle Brassica napus Mutant Cr are 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 Figure 1, provided in an embodiment of the present invention prominent using CRISPR/CAS9 technology acquisition cracking resistance angle cabbage type rape The method of variant obtains mutant single plant by genetic transformation using the homologous gene of CRISPR/Cas9 technology targeting BnIND, It is separated by selfing, finally obtains Mutants homozygous；And pass through the mutation type surface identification to acquisition, the measurement of cracking resistance angle property and something lost Pass analysis.

It specifically includes:

Vector construction: S101 targets bHLH structure using four sgRNA of CRISPR-P programming, first three sgRNA The upstream in domain, the targeting sequence of the 4th sgRNA is in bHLH structural domain.It is targeted with pYLCRIPSR/Cas9 multiple gene group Carrier system carries out vector construction, the carrier constructed by sequence verification.

Genetic transformation: S102 has separated the gene of BnA03.IND and BnC03.IND from cabbage type rape pure lines J9707 Group DNA and coded sequence, are suitable for Agrobacterium-medialed transformation.Carrier is turned with the hypocotyl genetic transformation of mediated by agriculture bacillus Enter semi-winterness cabbage type rape pure lines J9707 in, by the tissue-cultured seedling obtained after hygromycin selection be placed in a greenhouse growth ( Illumination/dark 16/8 hour at 22 DEG C).

Positive identification: S103 carries out PCR to the tissue-cultured seedling in greenhouse and confirms its positive, using specific primer to progress The presence of PCR amplification verifying T-DNA.

S104, editor's detection: positive using the method preliminary screening T0 generation based on polyacrylamide gel electrophoresis (PAGE) The mutation of genetically modified plants carries out PCR fragment direct Sequencing to editor's single plant after PAGE glue screening or is cloned into pEASY- In T1 carrier, the genotype for determining transgenic plant is sequenced using Sanger.

S105, selfing are homozygous: the T0 generation editor single plant self-pollination of acquisition generates T1 generation and T2 generation, near target site PCR product be sequenced to obtain double Mutants homozygous, these double Mutants homozygous can all cause frameshift mutation to be produced without function Protein.Double Mutants homozygous of a batch without T-DNA insertion are obtained by PCR verifying.

Gene expression amount analysis: S106 extracts the RNA of the different tissues in J9707, is carried out with the CDNA that RNA is inverted to QRT-PCR is tested to detect the expression of BnA03.IND and BnC03.IND in different tissues.

The measurement of cracking resistance angle: S107 carries out the survey of cracking resistance angle to the mutant and wild type of acquisition using the method for random collision It is fixed.20 complete siliques are put into cylindrical beaker (internal diameter 13.4cm, height 20.5cm), by 13 14 millimeters of diameter Steel ball put into beaker, be to be shaken 10 minutes on 300 revs/min of shaking table in revolving speed, cracking angle in every two minutes record beakers The number of fruit.According to cracking resistance angle calculation formulaCalculate cracking resistance ascent.

S108, Phenotypic Observation: marking in florescence and spend, and about 5 weeks collection siliques of Post flowering are used for microexamination.By silique Middle part cut 5mm length and fix and be embedded in paraffin.The cross section of 8 μ m-thicks is obtained using 2016 slicer of Leica RM. Lignin analysis is carried out to cross section, 2% phloroglucin of slice is handled 2 minutes, then develops the color and takes pictures in 50% hydrochloric acid. Image is obtained using Nikon ECLIPSE 80i compound microscope.It uses zoom stereoscope (SMZ-U, Nikon, Japan) It observes the form of complete mature silique and takes pictures.

In step S103, positive identification primer PB-R are as follows:

GCGCGCggtctcTACCGACGCGTATCC SEQ ID NO:1.

BnINDS3-F

AttGTGGCTTAGGGTTTCGGAAGG SEQ ID NO:2.

In step S104, editor's identification primer are as follows:

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, gene cloning primer BnIND-2 GGTCTATGCGTCTCTAGTCCAA SEQ ID NO:10.

BnIND-17 CAACATGAAACGCGTGATAGAA SEQ ID NO:11.

In the measurement of step S106 cracking resistance angle, the mutant of acquisition are as follows:

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 genomic DNA that step S102 is isolated, the nucleotide sequence of BnIND gene are as follows:

>BnA03.IND

ATGTCTGGCTCAAAAGCAGATGCAGCCATAGCCCCAATAGTCATGATGGAGCATCATCATCTCCTTAT GAATTGGAACAAACCTATTGATCTCATTACAGAAGAAAACTCTTTTAACCACAATCCTCATTTCATAGTAGATCCA CCTTCCGAAACCCTAAGCCACTTCCAGCCCCCGCCGACAATCTTCTCCGATCACGGAGGAGGAGAGGAAGCAGAAG AAGAAGAAGAAGAAGAAGGAGAGGAAGAGATGGATCCGATGAAGAAGATGCAATACGCGATTGCTGCCATGCAGCC CGTAGACCTCGATCCAGCCACCGTTCCTAAGCCGAACCGCCGTAACGTAAGGGTAAGCGACGACCCTCAGACGGTG GTGGCTCGTCGGCGTAGAGAAAGGATAAGCGAGAAGATCCGGATATTGAAGAGGATGGTGCCAGGCGGTGCAAAGA TGGACACTGCCTCCATGCTCGACGAAGCCATCCGCTACACCAAGTTCTTGAAACGGCAGGTGAGGCTAGCTTCTTC AGCCTCACACTCAGCTTGGAGCTCCTATGTCTGA SEQ ID NO:12.

>BnC03.IND

ATGTCTGGTTCAAAAGCAGATGCAGCAGCCATAGCTCCAATAGTCATGATGGAGCCTCATCATCTCCT TATGAACTGGAACAAACCTATTGATCTCATTACACAAGAAAACTCTTTTAACCACAATCCTCATTTCATGGTAGAT CCACCTTCCGAAACCCTAAGCCACTTCCAGCCCCCGCCGACAGTCTTCTCCGATCACGGAGGAGGAGAGGAAGCAG AAGACGAAGAAGGAGAGGAAGAGATGGATGAGATGAAGGAGATGCAATACGCGATTGCTGCCATGCAGCCCGTAGA CATCGATCCAGCCACCGTTCCTAAGCCGAACCGCCGTAACGTAAGGGTAAGCGAGGACCCCCAGACGGTGGTGGCT CGTCGGCGTAGAGAAAGGATAAGCGAGAAGATCCGGATATTGAAGAGGATGGTGCCAGGCGGTGCAAAGATGGACA CTGCCTCCATGCTCGACGAAGCCATCCGCTACACCAAGTTCTTGAAACGGCAGGTGAGGCTTCTTCAGCCTCACAC TCAGCTTGGGGCTCCTATGTCTGACCCTTCTTGCCTTTGTTATTACCACAACTCGG ATACCTAA SEQ ID NO: 13.

The invention will be further described combined with specific embodiments below.

Embodiment

The acquisition methods of cabbage type rape cracking resistance provided in an embodiment of the present invention angle gene BnIND function include:

(1), gene cloning: (seed comes from Wuhan, China rapeseed national project to plantation semi-winterness rape pure lines J9707 Research center), genomic DNA is extracted from fresh and tender blade, specific preparation method effectively extracts rape leaf total DNA referring to a kind of Method, Hua Zhong Agriculture University's journal, 1994,13 (5): 521-523, the method for report carry out, with 1% Ago-Gel electricity Swimming detection DNA mass, and with UV spectrophotometer measuring DNA concentration.Clone and separate obtains from the DNA of extraction The genomic DNA and coded sequence of BnA03.IND and BnC03.IND.

(2), vector construction: genomic DNA and coded sequence to isolated BnA03.IND and BnC03.IND into Row analysis devises four sgRNA on two copies of BnIND using CRISPR-P program.First three sgRNA targets bHLH The upstream of structural domain, the targeting sequence of the 4th sgRNA is in bHLH structural domain.Four sgRNA two copy in only There are the variation containing a base in BnA03.IND copy, other three sgRNA on first sgRNA to go up in two copies It is complete the same.Vector construction is carried out with pYLCRIPSR/Cas9 multiple gene group target carrier system, is constructed by sequence verification Carrier；

(3), the carrier built is transferred to semi-winterness Wild cabbage type oil with the hypocotyl genetic transforming method of mediated by agriculture bacillus Dish is sheerly in J9707, is carried out:

1) it sterilizes:

A. seed fills small semicanal with 2ml centrifuge tube, and 75% alcohol is added and impregnates seed 3min, notices that the time cannot be too long, Not so seed is not easy to germinate.

B. remove alcohol, 84 thimerosals (diluting one times with distilled water) are added and impregnate seed 3min；Remove 84 thimerosals, uses Aseptic water washing 4 to 5 times.Note: 84 thimerosal concentration sterile waters: commercialization 84 liquid=1:1；How much dosage is according to sub determine of committing genocide It is fixed, to be totally submerged seed.

2) sow: a. is multicast to M0 with the aseptic nipper seed that will sterilize, and every ware 10-12.B. culture dish is put into sterile culture In box, sets and cultivated 6 days at 24 DEG C of half-light.

3) bacterium is shaken: with LB training objective agrobacterium strains after sowing 4 days.Sterilize PU bottles of addition 4ml LB culture solutions, then inhales Enter the target bacteria of 10ul activation, and antibiotic is added, is i.e. 4ml LB liquid+4uL kan+4uL Gent+10uL bacterium night；In 28 Bacterium, about 15h are shaken in DEG C 180-220rpm shaking table.Note: it has to carry out object bacteria positive detection before shaking bacterium.- 80 DEG C of picking guarantors The agrobacterium strains deposited, in LB (anti-kanamycins and gentamicin) plate streaking culture, room temperature (28 DEG C or so) dark culture 40- 48h；Shake the survey of bacterium frontier inspection in picking single colonie side: a shakes bacterium: numbering and half spot of picking is seeded in the liquid LB that 1mL contains two kinds of antibiotic In, 28 DEG C, 220r/min culture.B detection: other half spot of picking 0.02M NaOH is cracked into 10min, utilizes special primer PCR detection is carried out, and negative and positive control is set, positive bacterium colony is chosen according to result.

4) it the preparation of explant and infects:

The OD value (in LB 0.8 or so preferably, general 16 hours) for surveying bacterium, draw the cultured bacterial strain of 2ml to 2ml without In bacterium centrifuge tube, 6000rpm is centrifuged 3min, outwells supernatant；It is suspended again with 2mlDM primary, 6000rpm is centrifuged 3min, in abandoning After clear, then 2mlDM suspends, by the bacterium solution to have suspended put 4 DEG C it is spare.

Seedling hypocotyl is equipped with 18ml M1 liquid during plate is prior into sterilized petri dishes after being cut sowing 6 days with sterile scissors Culture medium, the explant body length cut are 0.8-1.0cm, are disposably vertically cut as far as possible when cutting explant.Every ware 150-200 When a explant, the ready 2ml DM suspension bacteria liquid of the first step is added, starting timing dip dyeing 15min, (time cannot be grown, not so Explant is easily dead), the compartment time rocks once, and 4-5 times.Then bacterium solution is quickly sucked out, explant is transferred to sterile Pad have three layers filter paper plate in, siphon away a large amount of bacterium solutions adhered on explant.

5) it goes in M1 culture medium, the lower 24 DEG C of placements of 20-25 explant half-light of every ware.

6) it is gone in M2 after 2-3 days, and goes in light culture (24 DEG C daytime 16hs/ evening 8hs).

7) it is gone in M3 after 3 weeks, every 2-3 week subculture is primary, until there is green bud.

8) it is transferred to M4 root media to take root, rootage duration needs 2-4 weeks.Growing can be children after healthy and strong whole seedlings Seedling is transferred to crop field or greenhouse normal growth is solid.

The tissue-cultured seedling obtained after hygromycin selection is placed in a greenhouse growth, and (illumination/dark 16/8 is small at 22 DEG C When)；

(4), the detection of mutant:

(1) positive identification for carrying out transgenosis to mutation single plant with specific primer BnINDS3-F/PB-R, picks out and contains The positive single plant for thering is T-DNA to be inserted into.

(2) according to target proximity sequence, according to the primer premier5 design primer of the sequence near target fragment, Primer carries out blast analysis after determining, guarantees without other homologous sequences.

(3) PCR amplification is carried out with the target fragment primer of design.

(4) 1% Agarose horizontal electrophoresis detect PCR amplification effect.

(5) Native PAGE gel electrophoresis method detects pcr amplification product.

(6) PCR amplification is carried out to editor's single plant after PAGE glue screening, product is directly sent company, surveyed using Sanger Sequence determines the genotype of transgenic plant.

(5), selfing is homozygous: the T0 generation editor single plant self-pollination of acquisition generates T1 generation and T2 generation, near target site PCR product be sequenced to obtain the homozygous mutant of the homozygous and double copy of single copy, these Mutants homozygous can all cause frameshit prominent Sell of one's property the protein that raw function is lost.Double Mutants homozygous of a batch without T-DNA insertion are obtained by PCR sequence verification；

(6), the expression analysis of gene: extract J9707 in different tissues RNA, with rna transcription at CDNA carry out QRT-PCR experiment finds that two copy expression quantity in vegetative reproduction organ are higher, the expression quantity highest in silique skin, simultaneously The expression of BnA03.IND is significantly larger than the expression of BnC03.IND.

(7), cracking resistance angle measures: carrying out the survey of cracking resistance angle to the mutant and wild type of acquisition using the method for random collision It is fixed.20 complete siliques are put into cylindrical beaker (internal diameter 13.4cm, height 20.5cm), by 13 14 millimeters of diameter Steel ball put into beaker, be to be shaken 10 minutes on 300 revs/min of shaking table in revolving speed, cracking angle in every two minutes record beakers The number of fruit.Cracking resistance ascent is calculated according to cracking resistance angle calculation formula.As a result cracking resistance angle system of double Mutants homozygous than wild type Number increases about 1.3 times, has had reached the level of signifiance.

(8), Phenotypic Observation: marking in florescence and spend, and about 5 weeks collection siliques of Post flowering are used for microexamination.As a result table Bright, the fruit lobe marginal texture of double-mutant silique is obvious and wild type is different.It is fixed simultaneously that 5mm length will be cut in the middle part of silique It is embedded in paraffin.The cross section of 8 μ m-thicks is obtained using 2016 slicer of Leica RM.Lignin analysis is carried out to cross section, 2% phloroglucin of slice is handled 2 minutes, then develops the color and takes pictures in 50% hydrochloric acid.It is multiple using Nikon ECLIPSE 80i It closes microscope and obtains image.Use the form of zoom stereoscope (SMZ-U, Nikon, Japan) observation complete mature silique And it takes pictures.Mutant and wild type silique to acquisition carry out microexamination discovery, in interior peel edge, the mutant of BnIND Most middle pericarp membranes has the cell wall thicker than wild type.It there's almost no the wooden of peel edge in double-mutant Change layer and separating layer, b layers of the endocarp of lignifying are directly connected with the lignifying vascular bundle of diaphragm, form around pericarp Continuous lignifying group prevents the cracking of silique.Although the lignifying structure of single mutant is similar with wild type, there is also Some small differences.It is compared with wild type, single mutant shows one layer small of the non-wood keratinocyte cells to form separating layer.It removes Except this, in the mono- Mutants homozygous of BnA03.IND, pericarp and diaphragm are closely joined together by lignifying bridge and prevent angle The cracking of fruit.But in wild type and BnC03.IND single mutant, in the endocarp layer of lignifying and the diaphragm dimension of lignifying There are apparent separating layers among tubing.

The mono- Mutants homozygous of BnA03.IND and the extremely significant increase of double Mutants homozygous cracking resistance ascents.Sequence comparing analysis It was found that two copies of BnIND gene encode albumen with the same function, and promoter region has biggish sequence variations； The expression quantity that further gene expression analysis confirms that BnA03.IND is copied during Rape pod development is significantly higher than BnC03.IND copy.Thus, it could be seen that since the sequence difference of promoter region causes two copies of BnIND gene to occur Difference on gene expression amount eventually leads to BnA03.IND copy and sends out in siliqua of oilseed rape dehiscence process compared with BnC03.IND copy Wave bigger effect.

The invention will be further described combined with specific embodiments below.

Fig. 2 is the gene structure figure and carrier figure of BnIND two copies provided in an embodiment of the present invention.

In figure: (1) it includes an exon that white box, which represents the gene, and gray area indicates the bHLH structure of the gene Domain, the vertical line in genetic model indicate that target site, arrow indicate the direction of sgRNA.S1-S4 illustrates target sequence, leukorrhagia The area PAM of scribing line.(2) structure figures of SBnIND carrier.

Fig. 3 is the gene expression figure provided in an embodiment of the present invention in J9707 in different tissues.

Fig. 4 is the measurement result of cracking resistance ascent provided in an embodiment of the present invention, shows the mono- Mutants homozygous of BnA03.IND It is significantly higher than wild type figure with the cracking resistance ascent of double Mutants homozygous.

Fig. 5 is the phenotypic map provided in an embodiment of the present invention for indicating wild type and mutant.

Fig. 6 is that the embodiment of the present invention is provided in 18 periods of Rape pod development, the slice microexamination figure of silique.

A in figure, b, c, d are the structural schematic diagram before phloroglucinol stain；E, f, g, h are paraffin section phloroglucinol stain Effect picture afterwards, BnIND gene mutation keep silique fruit lobe margin development abnormal.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.

Sequence table

<110>Hua Zhong Agriculture University

<120>function and application of cabbage type rape cracking resistance angle gene BnIND

<160> 28

<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> 40

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 28

ccatccgcta caccaagttc ttgtggacac caagttcttg 40

Claims (8)

1. a kind of cabbage type rape cracking resistance angle gene BnIND, which is characterized in that cabbage type rape cracking resistance angle gene BnIND Nucleotides sequence is classified as SEQ ID NO:12, SEQ ID NO:13.

2. a kind of albumen of cabbage type rape cracking resistance as described in claim 1 angle gene BnIND coding, which is characterized in that described The amino acid sequence of albumen are as follows: SEQ ID NO:24, SEQ ID NO:25.

3. the control cabbage type rape cracking resistance that a kind of separation claim 1 requires cabbage type rape cracking resistance angle gene BnIND The promoter at angle, which is characterized in that the nucleotides sequence of the sequence promoter is classified as SEQ ID NO:26, SEQ ID NO:27.

4. a kind of utilize the double-mutant that cabbage type rape cracking resistance angle gene BnIND is mutated described in claim 1, feature It is, the double-mutant copies while occurring the nucleotide sequence mutation in gene coding region by two of BnIND, containing The plant for stating mutation has cracking resistance angle character, the double-mutant nucleotide sequence of cracking resistance angle phenotype is generated 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.

5. a kind of using the single mutation that cabbage type rape cracking resistance angle gene BnIND is mutated described in claim 1, feature exists In by the single copy of BnIND the nucleotide sequence mutation in gene coding region occurs for the single mutation, contains nucleosides after mutation The plant of acid sequence has cracking resistance angle character, and the single mutation nucleotides sequence that cracking resistance angle phenotype is generated after mutation is classified as SEQ ID NO: 28。

6. a kind of application using double mutation as claimed in claim 4 in breeding.

7. a kind of application of single mutation using described in claim 5 in breeding.

8. a kind of utilize cabbage type rape cracking resistance angle candidate gene BnIND described in claim 1 in cabbage type rape cracking resistance angle property The application of shape improvement kind.