CN110295178A - The expression for striking drop cabbage type rape and its parent species MYB43 is improving the application in disease resistance of plant - Google Patents

Abstract

The invention discloses strike drop MYB43 to improve the application in cabbage type rape and its parent species disease resistance.Cabbage type rape MYB43 gene family includes 4 member genes: BnMYB43-1, BnMYB43-2, BnMYB43-3 and BnMYB43-4, parent species Chinese cabbage, wild cabbage respectively have 2 member genes: BrMYB43-1, BrMYB43-2, BoMYB43-1 and BoMYB43-2.Plant expression vector is interfered by building RNA, is converted in cabbage type rape after double No. 10, transgenic positive plant stalk is obtained and is inoculated with sclerotinite, the scab that transgenosis strikes drop plant as the result is shown significantly becomes smaller, and disease resistance greatly improves；Transgenic plant plant type is smaller, but the yield of land area of one unit rises.The present invention is that MYB43 provides a kind of new method in terms of improving the disease resistance and dense planting high yield of cabbage type rape and its parent species.

Description

The expression for striking drop cabbage type rape and its parent species MYB43 is improving plant disease-resistant Application in property

Technical field

The present invention relates to gene engineering technology fields, in particular to strike drop MYB43 and improving cabbage type rape and its parent Application in species disease resistance.

Background technique

Cabbage type rape (Brassica napus, abbreviation rape) belongs to Cruciferae Brassica genus, is the Chinese cabbage by belonging to (Brassica rapa) doubles to be formed with wild cabbage (Brassica oleracea) by inter-species natural hybrization and genome Allotetraploid species.Due to natural hybrization and its subsequent at kind of process, only millennia, full-length genome are surveyed Sequence etc. is studies have shown that rape gathers around the sum of a full set of genome there are two parent species (Chinese cabbage, wild cabbage), and exists with parent species Corresponding intergenic sequence is almost consistent or height is similar.Rape and its parent species Chinese cabbage and wild cabbage are global important oil Material, vegetables, feed and ornamental plant.Worldwide, the Brassica genus such as rape, Chinese cabbage, wild cabbage or even entire Cruciferae are made In the production of object, by the harm of a variety of biologies and inanimate environment stress, wherein sclerotiniose occurs universal, and harm is huge, Sclerotiniose causes yield of rape to lose 10%-80%.

Myb transcription factor superfamily is of great significance for plant growth and development and adaptability, but overwhelming majority MYB base The Unknown Function of cause.For MYB43, it is special to pertain only to its a small amount of expression for having been reported in cruciate flower mesh plant at present Reference breath, AtMYB43 is expressed in the fiber of development in arabidopsis, and AtMYB43 promoter can be by dimension pipe developmental regulation The combination of factors A tMYB46.In the plant other than cruciate flower mesh, poplar PtrMYB152 is the homologous base of arabidopsis AtMYB43 Cause thickens stalk xylem after its overexpression, and secondary cell wall thickeies, and content of lignin increases.

But in entire plant kingdom so far, still shortage MYB43 and plant height, branch, plant type, lodging resistance, seed are produced The research of relationship between the characters such as amount, plant disease resistance is reported, the report of its molecular breeding application aspect is more lacked.For packet For the MYB43 gene for including the crucifer including arabidopsis, rape, Chinese cabbage, wild cabbage, has no genetically modified plants function and grind The report studied carefully.

Summary of the invention

In view of this, one of the objects of the present invention is to provide the tables for striking drop cabbage type rape and its parent species MYB43 Up to improving the application in disease resistance of plant, the second purpose, which is to provide, strikes drop cabbage type rape and its parent species MYB43 It expresses and is reducing the application in cabbage type rape or its parent species stem sclerotinite scab length；The third object of the present invention exists Cabbage type rape or its parent species unit plane are being improved in providing the expression for striking drop cabbage type rape and its parent species MYB43 Application in product yield.

In order to achieve the above objectives, the invention provides the following technical scheme:

1, the expression for striking drop cabbage type rape and its parent species MYB43 is improving the application in disease resistance of plant, described The full length cDNA sequence of cabbage type rape MYB43 family be SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27 and Shown in SEQ ID NO.28, the full length cDNA sequence of parent species Chinese cabbage MYB43 family is SEQ ID NO.29, SEQ ID Shown in NO.30, the full length cDNA sequence of parent species wild cabbage MYB43 family is shown in SEQ ID NO.31, SEQ ID NO.32.

Preferably, the overall length gDNA sequence of cabbage type rape MYB43 family is SEQ ID NO.33, SEQ ID Shown in NO.34, SEQ ID NO.35 and SEQ ID NO.36, the overall length gDNA sequence of parent species Chinese cabbage MYB43 family is SEQ Shown in ID NO.37, SEQ ID NO.38, the overall length gDNA sequence of parent species wild cabbage MYB43 family be SEQ ID NO.39, Shown in SEQ ID NO.40.

Preferably, the method for striking drop MYB43 is, for cabbage type rape, Chinese cabbage, wild cabbage MYB43 gene family, building Then the transformant of acquisition is converted cabbage type rape or its parent species by rna interference vector, obtain the plant for striking drop MYB43.

Preferably, rna interference vector 602-1081bp as shown in SEQ ID NO.25, shown in SEQ ID NO.26 604~1084bp, SEQ ID NO.27 shown in 606~1086bp, SEQ shown in 578~1058bp, SEQ ID No.28 602~1081bp shown in ID No.29,597 shown in 580~1060bp, SEQ ID No.31 shown in SEQ ID No.30 The CaMV35S of the antisense fragments insertion pFGC5941M carrier of 604~1084bp shown in~1077bp, SEQ ID No.32 is opened It is obtained between mover and BnPAP2 Intron II；

Or 602-1081bp shown in SEQ ID NO.25,604~1084bp, SEQ ID shown in SEQ ID NO.26 578~1058bp shown in NO.27,602 shown in 606~1086bp, SEQ ID No.29 shown in SEQ ID No.28~ 580~1060bp shown in 1081bp, SEQ ID No.30,597~1077bp, SEQ ID shown in SEQ ID No.31 The sense fragment of 604~1084bp shown in No.32 be inserted into pFGC5941M BnPAP2 Intron II and OCS 3 ' it Between, form RNAi carrier pFGC5941M-BMYB43I.

Preferably, the transformant is the Agrobacterium tumefaciems engineered strain containing the rna interference vector.

Preferably, shown plant is cabbage type rape, Chinese cabbage or wild cabbage.

2, the expression for striking drop cabbage type rape and its parent species MYB43 is reducing cabbage type rape or its parent species stem Application in bar sclerotinite scab length, the full length cDNA sequence of cabbage type rape MYB43 family be SEQ ID NO.25, Shown in SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, the full-length cDNA sequence of parent species Chinese cabbage MYB43 family It is classified as shown in SEQ ID NO.29, SEQ ID NO.30, the full length cDNA sequence of parent species wild cabbage MYB43 family is SEQ ID Shown in NO.31, SEQ ID NO.32.

3, the expression for striking drop cabbage type rape and its parent species MYB43 is improving cabbage type rape or its parent species list Application in the area output of position, the full length cDNA sequence of cabbage type rape MYB43 family are SEQ ID NO.25, SEQ ID Shown in NO.26, SEQ ID NO.27 and SEQ ID NO.28, the full length cDNA sequence of parent species Chinese cabbage MYB43 family is SEQ Shown in ID NO.29, SEQ ID NO.30, the full length cDNA sequence of parent species wild cabbage MYB43 family be SEQ ID NO.31, Shown in SEQ ID NO.32.

In the present invention, cabbage type rape explant kind is double tenth-seededs in commercial variety, and the explant is sterile Seedling hypocotyl section.

In the present invention, the index for improving cabbage type rape and its parent species disease resistance is to connect to transgenic plant stalk Kind sclerotinite, scab is significantly smaller than non-transgenic (control) plant, and disease resistance significantly improves.

In the present invention, changing Growth and development index is that plant is slightly short, and branch is less slightly slightly short, and blade is smaller, and single plant yield omits There is decline.But it can be resistant to dense planting, can increase the grain yield in land area of one unit after appropriate increase density.

The beneficial effects of the present invention are: the present invention provides in cabbage type rape and its parent species Chinese cabbage, wild cabbage Member gene's number, full length sequence, the coding protein specificity, evolutionary relationship of MYB43 family, and confirmed suitably to strike drop MYB43 man The expression of race can be such that the disease resistance of plant significantly increases, and yield increases after rational close planting.

Detailed description of the invention

In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out Illustrate:

Fig. 1 is the electrophoretogram (M:DNA of cabbage type rape, Chinese cabbage, wild cabbage MYB43 gene family full-length cDNA amplification marker；1~8 respectively represents BnMYB43-1, BnMYB43-2, BnMYB43-3, BnMYB43-4, BrMYB43-1, BrMYB43- 2, the PCR amplification band of the full-length cDNA of BoMYB43-1 and BoMYB43-2).

Fig. 2 be cabbage type rape, Chinese cabbage, wild cabbage MYB43 gene family overall length gDNA amplification electrophoretogram (M:DNA marker；1~8 respectively represents BnMYB43-1, BnMYB43-2, BnMYB43-3, BnMYB43-4, BrMYB43-1, BrMYB43- 2, the PCR amplification band of the overall length gDNA of BoMYB43-1 and BoMYB43-2).

Fig. 3 is the secondary structure of cabbage type rape, Chinese cabbage, wild cabbage MYB43 family member's albumen；Longitudinal 4 from long to short Kind line segment respectively indicates α spiral, extended chain, β-bend and random coils.The amino acid residue of digital representation albumen counting (A~ H successively represents BnMYB43-1, BnMYB43-2, BnMYB43-3, BnMYB43-4, BrMYB43-1, BrMYB43-2, BoMYB43- The secondary structure of 1 and BoMYB43-2 albumen).

Fig. 4 be cabbage type rape, Chinese cabbage, wild cabbage MYB43 family member's albumen tertiary structure (A~H is successively represented BnMYB43-1, BnMYB43-2, BnMYB43-3, BnMYB43-4, BrMYB43-1, BrMYB43-2, BoMYB43-1 and The tertiary structure of BoMYB43-2 albumen).

Fig. 5 is the system hair of cabbage type rape, Chinese cabbage, wild cabbage MYB43 family member and part arabidopsis myb transcription factor Raw relationship.

Fig. 6 is the organ specificity of cabbage type rape, Chinese cabbage, wild cabbage MYB43 family transcriptional expression.

Fig. 7 is the expression characteristic that cabbage type rape MYB43 family responds sclerotinite induction.

Fig. 8 is building (the A:RNA interference table of cabbage type rape and its parent species MYB43 family rnai expression carrier Up to electropherogram；B:RNA interference expression vector structure chart).

Fig. 9 is for the PCR identification of 3 optimal transgenic lines and its compared with adjoining tree expression, wherein 1,2,3 The transgenic line for striking drop MYB43 gene family is respectively indicated, the top (A) primer sets are combined into RBMYB43I+RBnPAP2I2, (A) Lower part primer sets are combined into FBMYB43I+ROCST5N, and primer sets are combined into FBMYB43RT+RBMYB43RT in (B).

Figure 10 is the variation of transgenic plant yield.

Figure 11 is the variation (A: stalk scab result figure of transgenic plant disease resistance；B: stalk scab length sequences).

Specific embodiment

Hereinafter reference will be made to the drawings, and a preferred embodiment of the present invention will be described in detail.It is not specified in preferred embodiment The experimental method of actual conditions, usually according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brooker Deng writing, Huang Peitang etc. translated, Science Press, 2002) described in condition, or according to the normal condition proposed by manufacturer.

The vegetable material that preferred embodiment uses: 821 (ZY821) of oil in cabbage type rape, in pairs No. 10 (ZS10) be Commercial variety, conventional field test condition plantation, bagging selfing are reserved seed for planting.

The reagent and kit that preferred embodiment uses: EASYspin plant RNA rapidly extracting kit is rich purchased from Beijing Mai De Bioisystech Co., Ltd；PrimeScript RT Reagent Kit with gDNA Eraser(Perfect Real Time) reverse transcription reagent box is purchased from Dalian TaKaRa company；Fluorescence quantitative kit FastStart Essential DNA Green Master is purchased from Roche company；Various restriction enzyme NcoI, AatII, BamHI and XbaI etc. are Lithuania MBI Fermentas Products；Murashige&Skoog (MS) culture medium is purchased from Duchefa company, Holland；GoldView nucleic acid dye Material is purchased from Beijing SBS Genetech gene technology Co., Ltd；CTAB, TE buffer, DNA Loading Buffer, rifampin (Rif), streptomysin (Str), kanamycins (Kan), ampicillin (Amp), Cefotaxime Sodium (Cef), agarose, tryptose Peptone, yeast extract, Basta herbicide and other reagents are purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

The key instrument that preferred embodiment uses: 9700 type PCR instrument of Applied Biosystems company, U.S. ABI, Bio-Rad CFX96 Touch^TMFluorescence quantitative PCR instrument；And molecular biology and plant genetic engineering other conventional instruments, Equipment and facility.

Embodiment 1, cabbage type rape, Chinese cabbage, wild cabbage MYB43 gene family clone

This research the primer is shown in Table 1.

Table 1, this research the primer

1, the extraction of cabbage type rape, Chinese cabbage, wild cabbage genome DNA and total serum IgE

Each strain takes the tender leaf of typical plant, and it is total to extract genome using cetyltrimethylammonium bromide (CTAB) method DNA, using the quality and concentration of 1.0% agarose gel electrophoresis method and spectrophotometry evaluation nucleic acid samples.Meanwhile with wild cabbage The root of type rape variety ZY821, stem, leaf, flower, the seed of Hua Hou 30d, Hua Hou 20d pod skin be material, it is a small amount of using pillar Plant total serum IgE extraction agent box by specification extracts total serum IgE, removes DNA impurity with DNase I, is dissolved in after total serum IgE ethanol precipitation Pure water.Nucleic acid samples detect quality using 1% agarose gel electrophoresis, are measured with Nanodrop 2000C ultraviolet specrophotometer Concentration and purity.

Electrophoresis result shows that the cabbage type rape extracted with CTAB method, Chinese cabbage, the integrality of wild cabbage genome DNA are good, Completely, the purity of spectrophotometry is also higher, can be used for PCR amplification and Southern hybrid experiment for RNA digestion.Electrophoresis point Analysis shows that the total serum IgE characteristic bands of acquisition are clear, no obvious RNA degradation and DNA pollution, the matter of spectrophotometry evaluation Amount is also preferable, can satisfy the requirement of downstream experiment.

2, the acquisition of total the first chain of cDNA of each organ of cabbage type rape, Chinese cabbage, wild cabbage

The PrimeScript RT Reagent Kit of TaKaRa company will be used after the RNA mixed in equal amounts mentioned in above-mentioned 1 With gDNA Eraser (Perfect Real Time) reverse transcription reagent box, obtains each device of cabbage type rape, Chinese cabbage, wild cabbage Total the first chain of cDNA of official.

3, the electronic cloning of cabbage type rape, Chinese cabbage, wild cabbage MYB43 gene family

Arabidopsis MYB43 gene (AtMYB43) be located at No. 5 chromosomes (AT5G16600) on, cDNA (AT5G16600) For 1329bp.Its cDNA sequence is submitted on the website NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) and is carried out Nucleic acid sequence alignment (nucleotide blast), inputted respectively in Organism Brassica napus (taxid: 3708), Brassica rapa (taxid:3711) and Brassica oleracea (taxid:3712), is selected in Database Others and Reference RNA sequence (refseq-rna) is selected, selects Somewhat in Optimize for Similar sequences (blatsn) then clicks BLAST and is compared, and comparison result shows in GenBank only two The DNA homolog score value for annotating into the gene order of BnMYB43 is higher, and 4 annotations are same at the gene of the sequence of other rapeseed genes Source score value is higher, and 2 annotations are higher at the DNA homolog score value of the sequence of other Chinese cabbage genes, and 2 annotations are at other wild cabbage bases The DNA homolog score value of the sequence of cause is higher, they are created sequence and is compared to arabidopsis website, finds this 10 sequences It is the orthologous genes of AtMYB43, they is subjected to multiple alignment discovery, they are divided into four groups.So speculating Wild cabbage type Rape, Chinese cabbage, wild cabbage are respectively present 4,2 and 2 MYB43 genes, and corresponding separate gene is respectively designated as BnMYB43- 1, BnMYB43-2, BnMYB43-3, BnMYB43-4, BrMYB43-1, BrMYB43-2, BoMYB43-1 and BoMYB43-2.

4, the clone of cabbage type rape, Chinese cabbage, wild cabbage MYB43 gene family full-length cDNA

Respectively using cabbage type rape, Chinese cabbage, total the first chain of cDNA of wild cabbage mixing organ as template, with primer listed by table 1 Combination carries out PCR amplification, and the agarose gel electrophoresis of pcr amplification product is shown, primer combines FBrMYB43-1+RBrMYB43- 1, FBrMYB43-2+RBrMYB43-2, FBoMYB43-1+RBoMYB43-1 and FBoMYB43-2+RBoMYB43-2 are amplified The band (Fig. 1) of 1 treaty 1200bp.After glue recycling, TA clone, the bacterium solution PCR detection of Batch Clone shows Insert Fragment There is polymorphism in length, equal sample presentation sequencing, the BLASTn of sequencing result and to show them with the comparison of AtMYB43 be sweet The MYB43 gene of blue type rape and its parent species, the length point for the standard full-length cDNA that the band sequencing of 8 1200bp obtains Not Wei 1235bp, 1236bp, 1199bp, 1217bp, 1235bp, 1202bp, 1242bp and 1215bp, be respectively designated as BnMYB43-1 mRNA、BnMYB43-2 mRNA、BnMYB43-3 mRNA、BnMYB43-4 mRNA、BrMYB43-1 mRNA、 BrMYB43-2 mRNA, BoMYB43-1 mRNA and BoMYB43-2 mRNA, nucleic acid sequence such as SEQ ID NO.25~SEQ Shown in ID NO.32.

5, cabbage type rape, Chinese cabbage, wild cabbage MYB43 gene family gDNA sequence clone

Template is substituted for cabbage type rape, Chinese cabbage, wild cabbage genome DNA, carries out PCR amplification as before, Electrophoresis result shows that primer combines FBrMYB43-1+RBrMYB43-1, FBrMYB43-2+RBrMYB43-2, FBoMYB43-1+ RBoMYB43-1 and FBoMYB43-2+RBoMYB43-2 has amplified the band (Fig. 2) of 1 treaty 2250bp.Glue recycling, TA grams After grand, bacterium solution PCR detects no polymorphism, be sequenced acquisition exact length be respectively 2293bp, 2252bp, 2237bp, 2256bp, 2295bp, 2240bp, 2259bp and 2258bp, be respectively designated as BnMYB43-1 gene, BnMYB43-2 gene, BnMYB43-3 gene, BnMYB43-4 gene, BrMYB43-1 gene, BrMYB43-2 gene, BoMYB43-1 gene and BoMYB43-2 gene, nucleic acid sequence is as shown in SEQ ID NO.33~SEQ ID NO.40.

Embodiment 2, cabbage type rape, Chinese cabbage, wild cabbage MYB43 gene family bioinformatic analysis

Sequence alignment, open reading frame (ORF) lookup and translation are carried out on Vector NTI Advance 11.5, The CDD search that BLAST and protein sequence are carried out on the website http://www.ncbi.nlm.nih.gov/, in http: // The websites such as bip.weizmann.ac.il/ and www.expasy.org provide and carry out protein on the bioinformatics website linked Structural analysis, in http://prodes.toulouse.inra.fr/multalin/multalin.html and http: // Gene and protein sequence multiple alignment and clustering are carried out on the websites such as www.ebi.ac.uk/clustalw/.

1, the structural analysis of cabbage type rape, Chinese cabbage, wild cabbage MYB43 gene family

(1) structural parameters of cabbage type rape, Chinese cabbage, wild cabbage MYB43 gene family

Cabbage type rape, Chinese cabbage, 8 full-length genes of wild cabbage MYB43 family DNA sequence dna be 2237-2295bp, have 3 A exon and 2 intrones, longest standard mRNA are 1242bp, and 5 ' noncoding region of longest (5 ' UTR) is 126bp, longest 3 ' UTR are 226bp, and coding amino acid is 304-329 (table 2).

Table 2, cabbage type rape, Chinese cabbage, wild cabbage MYB43 gene family structure and nucleic acid profile

(2) cabbage type rape, Chinese cabbage, the basic parameter of wild cabbage MYB43 family protein, subcellular localization and possible transcription After modify

8 cabbage type rape, Chinese cabbage, wild cabbage MYB43 family albumen of derivation have 304-329 amino acid residue, molecule Amount is 37.67-37.72kD, and isoelectric point is 6.9, and Charged acids account for 31.77-32.16%, and acidic amino acid accounts for 10.41- 10.42%, basic amino acid accounts for 11.38-11.39%, and polar amino acid accounts for 37.4-37.76%, and hydrophobic amino acid accounts for 26.68- 26.71%, be basic protein, with leucine content highest, followed by threonine in single amino acids, followed by asparagine or Serine.

SignalP5.0 predicts that they do not have signal peptide.Softberry-ProComp, WoLFPSORT and Plant-mPLoc Predict that they are positioned at nucleus；TMpred predicts that they do not have any transmembrane domain.It is multiple that NetPhos 3.1 predicts that they have Potential phosphorylation site, mainly Ser-phosphorylation site (table 3).

Table 3, cabbage type rape, Chinese cabbage, wild cabbage MYB43 family coding albumen essential characteristic

3 (Continued) of table

(3) conservative domain, conserved motifs and the higher structure of cabbage type rape, Chinese cabbage, wild cabbage MYB43 family protein

NCBI guards domain search (NCBI Conserved Domain Search) and shows cabbage type rape, Chinese cabbage, wild cabbage 8 albumen of MYB43 family are that SANT guards domain.

SOPMA software prediction, their secondary structure are mainly random coils (37.7-44.08%), followed by alpha-helix (33.2-36.4%) and extended chain (14.47-18.24%), β-corner (5.59-9.27%) (table 3, Fig. 3).

Swiss-Model predicts their gross three-dimensional structures (Fig. 4).

2, cabbage type rape, Chinese cabbage, wild cabbage MYB43 family homology and phylogenetic relationship

(1) homology of nucleic acid and protein level

Comparison two-by-two on Vector NTI Advance 11.5 shows cabbage type rape, Chinese cabbage, wild cabbage MYB43 family In the horizontal concordance rate of gDNA be 65.7%~99.6% between 8 full-length genes, the concordance rate of code area level for 83.1~ 99.9%, the consistency and similitude for encoding protein level are respectively 71.9~99.4% and 76%~99.7% (table 4).In core Sour water is flat, their yet further myb genes have certain homology.Their conservatives in code area are significantly higher than noncoding region Conservative, but introne in montage boundary be it is conservative, there is also the partial zones of some conservatives in 5 ' UTR and 3 ' UTR Domain, should be related with gene expression activity.In protein level, they also have certain homology (figure with many other MYB albumen 5)。

Table 4, cabbage type rape, Chinese cabbage, wild cabbage MYB43 family's nucleic acid and protein sequence homologies (%)

Note: gDNA concordance rate (italic, on), code area concordance rate (italic, under), albumen concordance rate (roman, on) and egg White likelihood (roman, under)

(2) phylogenetic relationship

What BLASTp was shown has the arabidopsis MYB albumen of similitude to download with cabbage type rape, Chinese cabbage, wild cabbage MYB43, Clustal W multiple ratio is utilized on Vector NTI Advance together with cabbage type rape, Chinese cabbage, wild cabbage MYB43 albumen Pair and construct genealogical tree (Fig. 5).Cabbage type rape, Chinese cabbage, wild cabbage MYB43 albumen and arabidopsis MYB43 are disclosed on genealogical tree Gather first for one kind, shows that cabbage type rape, Chinese cabbage, wild cabbage MYB43 albumen are the orthologous genes of AtMYB43 (orthologs)。

The relationship of embodiment 3, cabbage type rape MYB43 family's expression characteristic and anti-sclerotiniose

Select the pod of the root of oil 821, stem, leaf, flower, the seed of Hua Hou 30d, Hua Hou 20d in cabbage type rape commercial variety 6 histoorgans of pericarp and blade inoculation sclerotinite 0h, 0.5h, 3h, 9h, 2cm is organized around scab for 24 hours and after 48h For RNA sample as template, it is total for using PrimeScript RT Reagent Kit with gDNA Eraser reverse transcription CDNA, with the sense of organization specificity of fluorescence real-time quantitative PCR (qRT-PCR) analysis MYB43 gene family expression.It is combined with primer FBMYB43RT+RBMYB43RT detects the overall expression level of MYB43 gene in each organ.Primer combines FBrMYB43-1RT+ RBrMYB43-1RT, FBoMYB43-1RT+RBoMYB43-1RT, FBrMYB43-2RT+RBrMYB43-2RT and FBoMYB43- 2RT+RBoMYB43-2RT is respectively used to detection member gene BnMYB43-1, BnMYB43-2, BnMYB43-3 and BnMYB43-4 Expression.Reaction in 25 μ l standard FastStart Essential DNA Green Master-PCR systems into Row, the program of PCR reaction is all are as follows: 95 DEG C of initial denaturation 10min → 45 amplification cycles (95 DEG C of denaturation 10s → 60 DEG C/62 DEG C annealing 30s) → 65 DEG C to 95 DEG C of melting curve.

QRT-PCR the result shows that, BnMYB43 totality and its family member have expression in the above organ, but in stem, root Expression quantity be significantly larger than other organs (Fig. 6).In addition, the expression of BnMYB43 is lowered rapidly after being inoculated with sclerotinite 3h, explanation BnMYB43 participates in rape and sclerotinite Interaction (Fig. 7).

Embodiment 4 strikes the application of drop rape and its parent species MYB43 gene family in raising disease resistance

1, the clone of rape, Chinese cabbage, wild cabbage MYB43 gene family RNA interference fragment

Cabbage type rape ZY821 is used to mix total cDNA for template, using primer combination FBMYB43I+RBMYB43I amplification The interference fragment BMYB43I of BMYB43 gene family out, the product clip size of electrophoresis showed are identical as expection (Fig. 8, A).It will It is separately recovered, and connect with pMD 19-T, converts DH5 α, selects the monoclonal bacterium solution sequencing of the PCR positive, as the result is shown The length of BMYB43I is 505bp, 602-1081bp shown in respectively SEQ ID NO.25, shown in SEQ ID NO.26 578~1058bp shown in 604~1084bp, SEQ ID NO.27,606~1086bp, SEQ shown in SEQ ID No.28 602~1081bp shown in ID No.29,597 shown in 580~1060bp, SEQ ID No.31 shown in SEQ ID No.30 604~1084bp shown in~1077bp, SEQ ID No.32, without prominent compared with the full length cDNA sequence of the gene family Become.

2, the building of rape, Chinese cabbage, wild cabbage MYB43 gene family RNAi interference carrier

BMYB43I antisense fragments are subcloned into CaMV 35S in pFGC5941M using the complete double digestion of NcoI+AatII and open Between mover and BnPAP2 Intron II, BMYB43I sense fragment is subcloned into using the complete double digestion of BamHI+XbaI In pFGC5941M between BnPAP2 Intron II and OCS 3 ', formed RNAi carrier pFGC5941M-BMYB43I (Fig. 8, B). DH5 α is converted, the clone of anti-Kan is obtained, is combined through each primer carry out PCR detection respectively, positive clone molecule extracts plasmid After convert agrobacterium tumefaciens lba4404, PCR positive clone molecule is engineered strain.

3, mediated by agriculture bacillus pFGC5941M-BMYB43I converts rape commercial variety

In the present invention, it is used equally for improving after pFGC5941M-BMYB43I conversion rape, Chinese cabbage, wild cabbage and other plants Disease resistance, this preferred embodiment have selected conversion cabbage type rape as representing, convert the application principles of other species with it is such Seemingly.

All tissue cultures operations carry out under the conditions of the Plant Tissue Breeding of standard, between superclean bench, culture, tame and docile Clean rank between change is respectively 100 grades, 10000 grades and 100000 grades, and corresponding reagent, material, vessel press regulation and carry out nothing Bacterium processing.In after double tenth-seededs impregnate 1~2h in clear water, with after 75% ethyl alcohol surface sterilization 1min with aseptic water washing 3 It is secondary, 15min then is impregnated with 0.1% mercuric chloride, sterile water is repeatedly rinsed well, and MS solid medium [MS powder is then inoculated in 4.41g/L+Phytagel 2.6g/L+ sucrose 30.0mg/L, pH5.8, autoclave, which warms, to sterilize；It is liquid that Phytagel, which is not added, Body culture medium] on, 25 DEG C, 2000Lux illumination, 16h/d photoperiod cultivate that (condition of culture is except especially indicating person between subsequent tissue culture Outside, it is identical with this).The hypocotyl for cutting seedling age 8 days or so aseptic seedlings is cut into the segment for being about 0.5~1.0cm, is inoculated into pre- Train culture medium MSp [MS culture medium+1.0mg/L 6- benzylaminopurine (6-BA)+1.0mg/L 2,4 dichloro benzene ethoxyacetic acid (2,4-D)] on preculture 3 days.

The engineered strain of -80 DEG C of preservations is in the LB added with 100.0mg/L Kan+20.0mg/L Str+40.0mg/L Rif In 28 DEG C, 250r/min shaken cultivation 1~2 day in fluid nutrient medium, Agrobacterium is made to grow to logarithmic phase, switching culture is primary. Thalline were collected by centrifugation for 5000rpm, 10min room temperature, with dip dyeing culture medium MSm [MS fluid nutrient medium+1.0mg/L 2,4- dichloro-benzenes + 100 μM of acetosyringones (AS) of ethoxyacetic acid (2,4-D)+1.0mg/L 6- benzylaminopurine (6-BA)] adjust bacterial concentration To OD₆₀₀About 0.5 or so, as dip dyeing liquid for shell.

Hypocotyl section after preculture is immersed into 5-10min in dip dyeing liquid for shell, during which intermittence is gently swayed, then by small embryo Shaft part blots extra bacterium solution on sterilizing paper, is inoculated into total training culture medium MSc [MS solid medium+1.0mg/L 6-BA+ 1.0mg/L 2,4-D+50 μM of AS] in, 23.5 DEG C dark culture 48 hours.With sterilizing liquid culture medium MSk [MS fluid nutrient medium+ 1.0mg/L 2,4-D+1.0mg/L 6-BA+500mg/L cephalosporin (Cef)] washing by soaking 3 × 10min of explant, with sterilizing Paper blots surface liquid, is transferred to induction screening and culturing medium MSi [MS solid medium+1.0mg/L 6-BA+1.0mg/L2,4-D + 500mg/L Cef+15ppm Basta] in culture, about 2 weeks subcultures 1 time until growing macroscopic kanamycin-resistant callus tissue, then transfer To differential medium MSd [MS solid medium+4.0mg/L 6-BA+2.0mg/L zeatin (ZT)+5.0mg/L AgNO₃+ 500mg/L Cef+15ppm Basta] middle culture 14 days or more, evoked callus differentiates budlet, then is transferred to stem differentiation Culture in culture medium MSs (MS solid medium+3.0mg/L 6-BA+2.0mg/L ZT+500mg/L Cef+10ppm Basta) To growing small stem, then it is transferred to long shoot culture medium MSe (M solid medium+0.05mg/L 6-BA+500mg/L Cef+10ppm Basta it cultivates in) to the complete stem piece of length, then is transferred to root media MSr [MS solid medium+2mg/L methyl α-naphthyl acetate (NAA)] to flourishing root system is grown, the seedling after taking root is transplanted to after taming containing sterilizing perlite, vermiculite, grass for culture in In the basin alms bowl of charcoal soil (mass ratio 1:1:1) mixture, it is managed by greenhouse pot culture, finally obtains transgenic regenerated plant.

Meanwhile using double No. 10 regeneration plants in being obtained under the identical condition of tissue culture of no Basta screening pressure as non-transgenic Negative control.

4, the identification of transgenic plant

(1) Basta of transgenic plant rechecks identification

The Basta solution for being 200ppm with concentration apply at dye to the blade of transgenic regenerated plant and adjoining tree Reason.

(2) the PCR identification of transgenic plant

The blade of transgenic regenerated plant and negative control plant is taken respectively, genome DNA is extracted, using RBMYB43I This 2 pairs of primer combinations of+RBnPAP2I2 and FBMYB43I+ROCST5N carry out composite PCR detection.Positive transgenic regeneration plant exists Under the detection of 2 kinds of primers combination, it can expand and positive control (CK+, engineered strain) band of a size, and negative control Plant (WT) is then without band (Fig. 9).

5, the screening of the investigation of transgenic plant character and ideal strain

For transgenic plant compared with non-transgenic adjoining tree, some changes are had occurred in the agronomy form of transgenic plant Change: transgenic plant is slightly short, branch amount it is less slightly it is slightly short, blade is smaller, single plant yield is declined slightly, but properly increase planting density Yield goes up not down (Figure 10) afterwards.Stalk in the middle part of full-bloom stage is taken, counts stalk scab length after Vitro Inoculation Technique sclerotinite 96h.Root As a result, it has been found that, scab is significantly smaller (Figure 11) than non-transgenic (control) plant according to statistics.

It is detected, is found excessively short and small using expression of the qRT-PCR to MYB43 family in transgenic plant stem Strain in MYB43 family expression also very low i.e. RNA jamming effectiveness highest, and plant type is suppressed in the low plant of degree RNA jamming effectiveness is relatively low.On the contrary, qRT-PCR is also shown that the expression of MYB43 family and disease resistance are negatively correlated, i.e., The more high then disease resistance of RNA jamming effectiveness is stronger, and the more weak then disease resistance of RNA jamming effectiveness is weaker.It will be apparent that as a regulation base Cause, MYB43 are in the crossing on node of vine growth and development and disease resistance, and vine growth and development and disease resistance are two of balance Weights, and MYB43 is then the equalization point of balance.Then, the present invention comprehensively considered jamming effectiveness, disease resistance and plant type three it Between balance, from numerous transgenic lines find MYB43 family RNA jamming effectiveness it is significant in 20%-50% or so, disease resistance Enhancing, plant type slightly become smaller but the strain of energy dense planting volume increase is as ideal strain, and interfere excessively high and too low strain to give up those It abandons.

These results explanation can significantly improve the disease resistance of plant, slightly become smaller after suitably striking the expression for dropping MYB43 Plant type in the case where properly increasing planting density have crop field yield potential.

Other explanations of application form

1, the gene and its segment in the present invention also includes coming from other than nucleotide sequence listed in sequence table In the MYB43 gene order of rape, Chinese cabbage, other subspecies, the ecotype or the kind of wild cabbage and other nearly edge species, although it May have small difference with nucleotide sequence listed in sequence table.

2, the gene and its segment in the present invention, other than nucleotide sequence listed in sequence table, also include and it Have any nucleotide sequence of 98.00% or more consistency in continuous 80bp or more.

3, interference expression vector constructed in the present invention, in addition to the conversion pFGC5941M- as being lifted in preferred embodiments Other than BMYB43I, converting any one other carrier also can get similar effect.

4, the gene and its segment in the present invention, in addition to as lifted in preferred embodiments for cabbage type rape, Chinese cabbage, Other than wild cabbage, other species can also be applied to.

5, the gene and its segment in the present invention, in addition to disease-resistant using RNA interference enhancing as what is lifted in preferred embodiments Property and change plant type, similar effect can also be obtained using the technologies such as antisense RNA, CRES-T.

6, the gene and its segment in the present invention, in addition to being carried as what is lifted in preferred embodiments using pFGC5941M Other than body building, also to carry out vector construction using other platform carriers；Vector construct in the present invention, in addition to as preferential Other than the improvement leaf disk method mediated using agrobacterium tumefaciens lba4404 lifted in embodiment is converted, it can also be used Its method carries out Plant Transformation.

7, drop is struck by the MYB43 family in the present invention, neither from chromosome knock out all MYB43 genes completely, Nor the expression of entire MYB43 family is carried out 100% or the silencing close to 100%, although because such disease resistance Increase stronger, but growth and development is exceedingly inhibited, plant is excessively short and small and influences yield.The purpose of the present invention is suitably drop The expression of low MYB43 family, i.e. RNA jamming effectiveness is in 20%-50% or so.Therefore, obtained multiple independences are turned Gene strain needs the screening by qRT-PCR, identifies to disturb efficiency and suitably strike and drops strain as ideal strain, interference effect The too low and excessively high strain of rate is intended to give up.

Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention Protection scope within.Protection scope of the present invention is subject to claims.

Sequence table

<110>Southwest University

<120>expression for striking drop cabbage type rape and its parent species MYB43 is improving the application in disease resistance of plant

<160> 40

<170> SIPOSequenceListing 1.0

<210> 1

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

ggaaatacaa cattgtcagt atattag 27

<210> 2

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

atacgaggtt aaacagagaa aag 23

<210> 3

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

atgtcaaaat aaacagagaa aagtatg 27

<210> 4

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

atatacatta taacttgtaa gtatattagg tac 33

<210> 5

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gagaaaagta tgttcacaat tgcaata 27

<210> 6

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gagaaaagta tgttcacaat tgcaatc 27

<210> 7

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

gccaatgctg ttggagagc 19

<210> 8

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

tcagagagag gcttatgggt ca 22

<210> 9

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

agacggtgcg caacaacaag 20

<210> 10

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

cttgccgtcg ttgtaattaa tgttac 26

<210> 11

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

agaaggtgcg cagcaacaag 20

<210> 12

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

ctttccgtct ttgtaattaa tgtcac 26

<210> 13

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

actggacatc tcttttgctg at 22

<210> 14

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

cttgtcgtct ttgtaaataa tattatcct 29

<210> 15

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

actggacatc tcttttgctc ag 22

<210> 16

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

cttgtcgtct ttgtaaataa tattatccc 29

<210> 17

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

gatttctgcc cagtgctctg aa 22

<210> 18

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

tctgccaagc ccgttccctt 20

<210> 19

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

ggatccgacg tcaacaacat caactaggga aac 33

<210> 20

<211> 35

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

tctagaccat ggagaagatc aaatgtccat gaatc 35

<210> 21

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

ggaagttcat ttcatttgga gag 23

<210> 22

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

gctcaggttt tttacaacgt gcac 24

<210> 23

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 23

atttaaatga cgtcaggttt acattcaaga caca 34

<210> 24

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 24

cctaggtgga ttcgtacgta aactttt 27

<210> 25

<211> 1235

<212> DNA

<213>cabbage type rape (Brassica napus)

<400> 25

ggaaatacaa cattgtcagt atattagata tctatttgag cacatctcta gactatataa 60

agtaagaaaa atatatatag agagaggaga gagatgggga ggcaaccatg ttgtgacaaa 120

gtaggcttga agaaaggccc atggactgtt gaagaagaca agaagcttat caacttcatc 180

ctcaccaatg gccaatgctg ttggagagct cttcccaagc tttctggact attgaggtgt 240

ggtaaaagct gcagattaag atggataaat tatttgagac ctgatttaaa aagaggtctt 300

ttatcagaaa atgaagagca gatggtcatt gatcttcatg cccaacttgg caatagatgg 360

tcaaagattg cctctcatct accaggaaga actgataatg aaattaagaa ccattggaac 420

acacacataa agaaaaaact aaggaagatg ggtattgacc ctatgaccca taagcctctc 480

tctgaacaag acggtgcgca acaacaagct caggggagaa agaaaagctt agtgtctcgt 540

gatgagaaga acacaaacca aggccaagaa gatcaacaac aaactagaaa agaagaagat 600

caacaacatc aactagggaa agatttggag aagaacaaca cacccatttc tactgatgat 660

gggttttgca ttgatgatat cccattactc agtccacatg agatatttgt ggacatccct 720

tctgctcacc atcatactag tgatgataat gccaacatta acattagtaa ctccacttca 780

ccttcttcgt cttcctcttc tacctcgtcg tgcatatcat cactaccggg tgatgagttc 840

tcaaagtttc ttgatgaaat ggagattgtt gacctcaagt ggctctcatc cgatgattca 900

tcagggggta acattaatta caacgacggc aagttcaaca acaatgttga tacgatgagc 960

ttgtgggaca tcaatgattt gagcagcttg gaattgttta tgaatgatca tgatgatagt 1020

gtggttgaag cggatgttca agaatggttt tagatcatga ttcatggaca tttgatcttc 1080

tttaactttt agtttttttt tcttctcatg ttgtttttaa ttgtttttgt ttgtttgttt 1140

gggtttggcc ttggtggtgg atgattaggt gtaaaagtac atatgtaatt aaagattgca 1200

attgtgaaca tacttttctc tgtttaacct cgtat 1235

<210> 26

<211> 1236

<212> DNA

<213>cabbage type rape (Brassica napus)

<400> 26

ggaaatacaa cattgtcagt atattagata tctattcgag cacatctcta gactatataa 60

agaaagaaaa atatatagag agagagagga gagagatggg gaggcaacca tgttgtgaca 120

aagtaggctt gaagaaaggg ccatggactg ttgaagaaga caagaagctc atcaacttca 180

tcctcaccaa tggccaatgc tgttggagag ctcttcccaa gctttctgga ctattgaggt 240

gtggtaaaag ctgcagatta agatggataa attatttgag acctgattta aaaagaggtc 300

ttttatctga aaatgaagag cagatggtca ttgatcttca tgcccaactt ggcaatagat 360

ggtcaaagat tgcctctcat ctaccaggaa gaactgataa tgaaattaag aaccattgga 420

acacacacat aaagaaaaaa ctaaggaaga tgggtattga ccctatgacc cataagcctc 480

tctctgaaca agacggtgcg caacaacaag ctcaggggat aaagaaaagc ttagtgtctc 540

gtgatgagaa gaacacaaac caaggccaag aagatcaaca acaaactaga aaagaagaag 600

atcaacaaca tcaactaggg aaagatttgg agaagaacaa cacacccatt tctactgatg 660

atgggttttg cattgatgat atcccattac tcagtccaca tgagatattt gtggacatct 720

cttctgctca ccatcatact agtgatgata atgccaacat taacattagt aactccactt 780

caccttcttc gtcttcctct tctacttcgt catgcatatc atcactaccg ggtgatgagt 840

tctcaaagtt tcttgatgaa atggagattg ttgatctcaa gtggctctca tccgatgatt 900

catcaggggg tgacattaat tacaacgacg gcaagttcaa caacaatgtt gatacgatga 960

gcttgtggga catcaatgat ttgagcagct tggaattgtt ttatgaatga tcatgatgat 1020

agtgtggttg aagcggatgt tcaagaatgg ttttagatca tgattcatgg acatttgatc 1080

ttctttaact tttagttttt tttttcttct cttgttgttt ttaattgttt ttgtttgttt 1140

gtttgggttt ggccttggtg ttgcagttta gtggatgatt aggtgtaaaa gtacatatgt 1200

aattaaagat tgcaattgtg aacatacctt tcactg 1236

<210> 27

<211> 1199

<212> DNA

<213>cabbage type rape (Brassica napus)

<400> 27

cttgtttgag catatctcta gactaatata tatatatata tatatataga gagagagaga 60

cagagggtgc gaggagtgaa gatggggagg cagccatgtt gtgacaaagt agggctgaag 120

aaagggccat ggacagctga ggaagacaag aagctcatca acttcatcct caccaatggc 180

cactgctgtt ggagagctct tcccaagctt tctggactct tgaggtgtgg aaaaagctgc 240

agactaagat ggatcaatta tttgagacct gatttaaaaa gaggtctttt atcagaatgt 300

gaagaacaga tggtcattga tcttcatgcc caacttggca atagatggtc aaagattgcc 360

tctcatctac caggaagaac tgataatgaa ataaagaacc attggaacac acacataaag 420

aaaaaactaa ggaagatggg cattgaccct atgacccata agcctctctc tgaacaagac 480

gggtcacagc aagctcaagg gagcaagagt ttagtgcctc atgatgacaa gaacaaaaaa 540

caagaccaag aagataaaca aactaaagaa aaacaagaac accatcaact agggatagat 600

ttggacaaga gcaacaaaac aatatctact gatgatgggt tttgcattga tgaagtcccg 660

ttgctcgatc cacatgagat attactggac atctcttttg ctgatcatca tactagtgat 720

gaaaatgctc acattaacat tagtaactcc acttctcctt cttcctcttc tacctcgtcg 780

tgtatatcat cagtaccggg tgaggagttc tcgaagtttc ttgatgaaat ggacactatt 840

gacctcaagt ggctctcatc cgatcattca tcagaggata atattattta caaagacgac 900

aagttccaca acaatgttga tacgatgaac ttgtgggaca taaattattt gagcagcttg 960

gaattgttta tgaatgatca tgatgatggt tttgttggaa gcggatgttc aagaatggtt 1020

tagatcaata tacatgacca ttttactttc agtttttttt tcatgttgtt tttaattagt 1080

tctttttgtt tgttttcgaa gtgggcgatt tggatttggc cttggtgttg caatttagtg 1140

gatgattagg tgtaaaatac atatgtaata aatattgcaa ttgtgaacac acttttctc 1199

<210> 28

<211> 1217

<212> DNA

<213>cabbage type rape (Brassica napus)

<400> 28

atatacatta taacttgtaa gtatattagg tacttgtttg agcatatctc tagactaata 60

gatatatata tatatagaga gagagagcca gagggtgcga ggagtgaaga tggggaggca 120

gccatgttgt gacaaagtag ggctgaagaa agggccatgg acagctgagg aagacaagaa 180

gctcatcaac ttcatcctca ccaatggcca ctgctgttgg agagctcttc ccaagctttc 240

tggactcttg aggtgtggaa aaagctgcag actaagatgg atcaattatt tgagacctga 300

tttaaaaaga ggtcttttat cagaatgtga agaacagatg gtcattgatc ttcatgccca 360

acttggcaat agatggtcaa agattgcctc tcatctacca ggaagaactg ataatgaaat 420

aaagaaccat tggaacacac acataaagaa aaaactaagg aagatgggca ttgaccctat 480

gacccataag cctctccctg aacaagacgg ttcacagcaa gctcaaggga gcaagagttt 540

agtgcctcat gatgacaaga acaaaaaaca agaccaagaa gataaacaaa ctaaagaaaa 600

acaagaacac catcaactag ggatagattt ggacaagagc aacaaaacaa tatctactga 660

tgatgggttt tgcattgatg aagtcccgtt gctcgatcca catgagatat tactggacat 720

ctcttttgct cagcatcata ctagtgatga aaatgctcac attaacatta gtaactccac 780

ttctccttct tcctcttcta cctcgtcgtg tatatcatca gtaccgggtg aggagttctc 840

gaagtttctt gatgaaatgg agactattga cctcaagtgg ctctcatccg atcattcatc 900

aggggataat attatttaca aagacgacaa gttccacaac aatgttgata cgatgaactt 960

gtgggacata aattatttga gcagcttgga attgtttatg aatgatcatg atgatggttt 1020

tgttggaagc ggatgttcaa gaatggttta gatcaatatt catgaccatt ttactttcag 1080

tttttttttt catgttgttt ttaattagtt ctttttgttt gttttcgaag tgggcgattc 1140

ggatttggcc gtggtgttgc aatttagtgg atgattaggt gtaaaataca tatgtaataa 1200

agattgcaat tgtgaac 1217

<210> 29

<211> 1235

<212> DNA

<213>Chinese cabbage (Brassica rapa)

<400> 29

ggaaatacaa cattgtcagt atattagata tctatttgag cacatctcta gactatataa 60

agtaagaaaa atatatatag agagaggaga gagatgggga ggcaaccatg ttgtgacaaa 120

gtaggcttga agaaaggccc atggactgtt gaagaagaca agaagcttat caacttcatc 180

ctcaccaatg gccaatgctg ttggagagct cttcccaagc tttctggact attgaggtgt 240

ggtaaaagct gcagattaag atggataaat tatttgagac ctgatttaaa aagaggtctt 300

ttatcagaaa atgaagagca gatggtcatt gatcttcatg cccaacttgg caatagatgg 360

tcaaagattg cctctcatct accaggaaga actgataatg aaattaagaa ccattggaac 420

acacacataa agaaaaaact aaggaagatg ggtattgacc ctatgaccca taagcctctc 480

tctgaacaag acggtgcgca acaacaagct caggggagaa agaaaagctt agtgtctcgt 540

gatgagaaga acacaaacca aggccaagaa gatcaacaac aaactagaaa agaagaagat 600

caacaacatc aactagggaa agatttggag aagaacaaca cacccatttc tactgatgat 660

gggttttgca ttgatgatat cccattactc agtccacatg agatatttgt ggacatctct 720

tctgctcacc atcatactag tgatgataat gccaacatta acattagtaa ctccacttca 780

ccttcttcgt cttcctcttc tatctcgtcg tgcatatcat cactaccggg tgatgagttc 840

tcaaagtttc ttgatgaaat ggagattgtt gacctcaagt ggctctcatc cgatgattca 900

tcagggggta acattaatta caacgacggc aagttcaaca acaatgttga tacgatgagc 960

ttgtgggaca tcaatgattt gagcagcttg gaattgttta tgaatgatca tgatgatagt 1020

gtggttgaag cggatgttca agaatggttt tagatcatga ttcatggaca tttgatcttc 1080

tttaactttt agtttttttt tcttctcatg ttgtttttaa ttgtttttgt ttgtttgttt 1140

gggtttggcc ttggtggtgg atgattaggt gtaaaagtac atatgtaatt aaagattgca 1200

attgtgaaca tacttttctc tgtttaacct cgtat 1235

<210> 30

<211> 1202

<212> DNA

<213>Chinese cabbage (Brassica rapa)

<400> 30

cttgtttgag catatctcta gactaatata tatatatata tatatatata tagagagaga 60

gacagagggt gcgaggagtg aagatgggga ggcagccatg ttgtgacaaa gtagggctga 120

agaaagggcc atggacagct gaggaagaca agaagctcat caacttcatc ctcaccaatg 180

gccactgctg ttggagagct cttcccaagc tttctggact cctgaggtgc ggaaaaagct 240

gcagactaag atggatcaat tatttgagac ctgatttaaa aagaggtctt ttatcagatt 300

gtgaagaaca gatggtcatt gatcttcatg cccaacttgg caatagatgg tcaaagattg 360

cctctcatct accaggaaga actgataatg aaataaagaa ccattggaac acacacataa 420

agaaaaaact aaggaagatg ggcattgacc ctatgaccca taagcctctc cctgaacaag 480

acgggtcaca gcaagctcaa gggagcaaga gtttagtgcc tcatgatgac aagaacaaaa 540

aacaagacca agaagataaa caaactaaag aaaaacaaga acaccatcaa ctagggatag 600

atttggacaa gagcaacaaa acaatatcta ctgatgatgg gttttgcatt gatgaagtcc 660

cgttgctcga tccacatgag atattactgg acatctcttt tgctgatcat catactagtg 720

atgaaaatgc tcacattaac attagtaact ccacttctcc ttcttcctct tctacctcgt 780

cgtgtatatc atcagtaccg ggtgaggagt tctcgaagtt tcttgatgaa atggacacta 840

ttgacctcaa gtggctctca tccgatcatt catcagagga taatattatt tacaaagacg 900

acaagttcca caacaatgtt gatacgatga acttgtggga cataaattat ttgagcagct 960

tggaattgtt tatgaatgat catgatgatg gttttgttgg aagcggatgt tcaagaatgg 1020

tttagatcaa tatacatgac cattttactt tcagtttttt ttttcatgtt gtttttaatt 1080

agttcttttt gtttgttttc gaagtgggcg atttggattt ggccttggtg ttgcaattta 1140

gtggatgatt aggtgtaaaa tacatatgta ataaatattg caattgtgaa catacttttc 1200

tc 1202

<210> 31

<211> 1242

<212> DNA

<213>wild cabbage (Brassica oleracea)

<400> 31

ggaaatacaa cattgtcagt atattagata tatttgagca catctctaga ctatataaag 60

taagaaaaat atatatagag agaggagaga gatggggagg cagccatgtt gtgacaaagt 120

aggcttgaag aaagggccat ggactgttga agaagacaag aagctcatca acttcatcct 180

caccaatggc caatgctgtt ggagagctct tcccaaactt tctggactat tgaggtgtgg 240

taaaagctgc agattaagat ggataaatta tttgagacct gatttaaaaa gaggtctttt 300

atcagaaaat gaagagcaga tgatcattga tcttcatgcc caacttggca atagatggtc 360

aaagattgcc tctcatctac caggaagaac tgataatgaa attaagaacc actggaacac 420

acacataaag aaaaaactaa ggaagatggg tattgaccct atgacccata agcctctctc 480

tgaacaagaa ggtgcgcagc aacaagctca ggggacaaag aaaagcttag tgtctcgtga 540

tgagaagaaa agaaaccaag gccaagaaga tcaacaaact agaaaagaag aagatcaaca 600

acatcaacta gggaaacatt tggacaagaa caacacaccc atttctactg atgatgggtt 660

ttgcattgat gatatcccat tactcaatcc acatgagata tttgtggaca tctcttctgc 720

tcaccatcat actagtgatg gtaatgccaa cattaacatt agtaactcca cttcaccatc 780

ttcgtcttcc tctcctacct cgtcgtgcat atcatcacta ccaggtgatg agttctcaaa 840

gtttcttgat gaaatggaga ttgttgacct caagtggctc tcatccgatg attcgtcagg 900

gggtgacatt aattacaaag acggaaagtt caataacaat gttgatacga tgaacttgtg 960

ggacatcaat gatttgagca gcttggaatt gtttatgaat gatcacgatg atagtgtggt 1020

tggaagcgga tgttcaagaa tggttttaga tcatgattca tggacatttg atcttcttta 1080

acttttagtt tttttttctt ctcttgttgt ttttaattgt tttttgtttg tttgtttggg 1140

tttggcctcg gtgttgcagt ttagtggatg attaggtgta aaagtacata tgtaattaaa 1200

gattacaatt gtgaacatac ttttctctgt ttattttgac at 1242

<210> 32

<211> 2430

<212> DNA

<213>wild cabbage (Brassica oleracea)

<400> 32

atatacatta taacttgtaa gtatattagg tacttgtttg agcatatctc tagactaata 60

gatatatata tagagagaga gagagccaga gggtgcgagg agtgaagatg gggaggcagc 120

catgttgtga caaagtaggg ctgaagaaag ggccatggac agctgaggaa gacaagaagc 180

tcatcaactt catcctcacc aatggccact gctgttggag agctcttccc aagctttctg 240

gactcttgag gtgtggaaaa agctgcagac taagatggat caattatttg agacctgatt 300

taaaaagagg tcttttatca gaatgtgaag aacagatggt cattgatctt catgcccaac 360

ttggcaatag atggtcaaag attgcctctc atctaccagg aagaactgat aatgaaataa 420

agaaccattg gaacacacac ataaagaaaa aactaaggaa gatgggcatt gaccctatga 480

cccataagcc tctccctgaa caagacggtt cacagcaagc tcaagggagc aagggtttag 540

tgcctcatga tgacaagaac aaaaaacaag accaagaaga taaacaaact aaagaaaaac 600

aagaacacca tcaactaggg atagatttgg acaagagcaa caaaacaata tctactgatg 660

atgggttttg cattgatgaa gtcccgttgc tcgatccaca tgagatatta ctggacatct 720

cttttgctca gcatcatact agtgatgaaa atgctcacat taacattagt aactccgctt 780

ctccttcttc ctcttctacc tcgtcgtgta tatcatcagt accgggtgag gagttctcga 840

agtttcttga tgaaatggag actattgacc tcaagtggct ctcatccgat cattcatcag 900

gggataatat tatttacaaa gacgacaagt tccacaacaa tgttgatacg atgaacttgt 960

gggacataaa ttatttgagc agcttggaat tgtttatgaa tgatcatgat gatggttttg 1020

ttggaagcgg atgttcaaga atggtttaga tcaatattca tgaccatttt actttcagtt 1080

ttttttttca tgttgttttt aattagttct ttttgtttgt tttcgaagtg ggcgatttgg 1140

atttggccgt ggtgttgcaa tttagtggat gattaggtgt aaaatacata tgtaataaag 1200

attgcaattg tgaacatata cattataact tgtaagtata ttaggtactt gtttgagcat 1260

atctctagac taatagatat atatatagag agagagagag ccagagggtg cgaggagtga 1320

agatggggag gcagccatgt tgtgacaaag tagggctgaa gaaagggcca tggacagctg 1380

aggaagacaa gaagctcatc aacttcatcc tcaccaatgg ccactgctgt tggagagctc 1440

ttcccaagct ttctggactc ttgaggtgtg gaaaaagctg cagactaaga tggatcaatt 1500

atttgagacc tgatttaaaa agaggtcttt tatcagaatg tgaagaacag atggtcattg 1560

atcttcatgc ccaacttggc aatagatggt caaagattgc ctctcatcta ccaggaagaa 1620

ctgataatga aataaagaac cattggaaca cacacataaa gaaaaaacta aggaagatgg 1680

gcattgaccc tatgacccat aagcctctcc ctgaacaaga cggttcacag caagctcaag 1740

ggagcaaggg tttagtgcct catgatgaca agaacaaaaa acaagaccaa gaagataaac 1800

aaactaaaga aaaacaagaa caccatcaac tagggataga tttggacaag agcaacaaaa 1860

caatatctac tgatgatggg ttttgcattg atgaagtccc gttgctcgat ccacatgaga 1920

tattactgga catctctttt gctcagcatc atactagtga tgaaaatgct cacattaaca 1980

ttagtaactc cgcttctcct tcttcctctt ctacctcgtc gtgtatatca tcagtaccgg 2040

gtgaggagtt ctcgaagttt cttgatgaaa tggagactat tgacctcaag tggctctcat 2100

ccgatcattc atcaggggat aatattattt acaaagacga caagttccac aacaatgttg 2160

atacgatgaa cttgtgggac ataaattatt tgagcagctt ggaattgttt atgaatgatc 2220

atgatgatgg ttttgttgga agcggatgtt caagaatggt ttagatcaat attcatgacc 2280

attttacttt cagttttttt tttcatgttg tttttaatta gttctttttg tttgttttcg 2340

aagtgggcga tttggatttg gccgtggtgt tgcaatttag tggatgatta ggtgtaaaat 2400

acatatgtaa taaagattgc aattgtgaac 2430

<210> 33

<211> 2293

<212> DNA

<213>cabbage type rape (Brassica napus)

<400> 33

ggaaatacaa cattgtcagt atattagata tctatttgag cacatctcta gactatataa 60

agtaagaaaa atatatatag agagaggaga gagatgggga ggcaaccatg ttgtgacaaa 120

gtaggcttga agaaaggccc atggactgtt gaagaagaca agaagcttat caacttcatc 180

ctcaccaatg gccaatgctg ttggagagct cttcccaagc tttctggtcg attttctctc 240

cttatttcct ccttaaacga catcttttgg gatttctcat gtgtaaaagt tcactttctt 300

gcttcttgtg tgtttagcct ttctctacgc caactgacgc ttttttggtg ctatttctct 360

acaccgacga aactgatgct attttgattt ttagatactc gtcttttaaa agttcacttc 420

cttaaaacgt ccaattttat tactagtaat ttctatttct ctactttctt cttgtcagca 480

gtttttatag taatattttc tctacttttg tgtaaattaa tttgcatgag ttattttggt 540

atatgcatgc tagtatagat gttcaccaac aatcaaatat acctttagaa attagtttca 600

agaaacaatt ttgatgcagg agaatcatat gaattcttaa ctttcctttt tgaaatacaa 660

tatatatttt aatatcggtt ggacctaaaa acaaaagtct caaaacatat aattagacta 720

ttaatcatag gctcatagcg tctggcccga aaagaaatat agtgcttgat attcgtggtc 780

ctcacacgtt attagttgat cttgataaaa aaaaattgtt ttgtttttgg ctttttgttt 840

agtgcttcct gccaaaatat atttcgtacg gttttaactt tcaatcaatc tgattgttaa 900

tttaattact ttggttaatt aactaaatgc aggactattg aggtgtggta aaagctgcag 960

attaagatgg ataaattatt tgagacctga tttaaaaaga ggtcttttat cagaaaatga 1020

agagcagatg gtcattgatc ttcatgccca acttggcaat aggtttatcc tctttttatt 1080

caaaataatt gttatttcat attataaaca taatttactt accttatttg acattgtaac 1140

taactcataa tataatgaaa tgtttaaaga ttatggacat tagatttcaa tattaaaggt 1200

attggagaag cacaaaagtc attatatgac gcaaacttta tcactttttt tgcatgaaag 1260

agattggcta atccacattt ttaattactc atattttctt tcaaacccaa tagtttaatt 1320

aagtcggtgt gatgaaaatg tttgaacagt cgaaatcatt agtgaaatta tcacttataa 1380

aattttcaaa tattttattt ttatttttgt gcagatggtc aaagattgcc tctcatctac 1440

caggaagaac tgataatgaa attaagaacc attggaacac acacataaag aaaaaactaa 1500

ggaagatggg tattgaccct atgacccata agcctctctc tgaacaagac ggtgcgcaac 1560

aacaagctca ggggagaaag aaaagcttag tgtctcgtga tgagaagaac acaaaccaag 1620

gccaagaaga tcaacaacaa actagaaaag aagaagatca acaacatcaa ctagggaaag 1680

atttggagaa gaacaacaca cccatttcta ctgatgatgg gttttgcatt gatgatatcc 1740

cattactcag tccacatgag atatttgtgg acatcccttc tgctcaccat catactagtg 1800

atgataatgc caacattaac attagtaact ccacttcacc ttcttcgtct tcctcttcta 1860

cctcgtcgtg catatcatca ctaccgggtg atgagttctc aaagtttctt gatgaaatgg 1920

agattgttga cctcaagtgg ctctcatccg atgattcatc agggggtaac attaattaca 1980

acgacggcaa gttcaacaac aatgttgata cgatgagctt gtgggacatc aatgatttga 2040

gcagcttgga attgtttatg aatgatcatg atgatagtgt ggttgaagcg gatgttcaag 2100

aatggtttta gatcatgatt catggacatt tgatcttctt taacttttag tttttttttc 2160

ttctcatgtt gtttttaatt gtttttgttt gtttgtttgg gtttggcctt ggtggtggat 2220

gattaggtgt aaaagtacat atgtaattaa agattgcaat tgtgaacata cttttctctg 2280

tttaacctcg tat 2293

<210> 34

<211> 2252

<212> DNA

<213>cabbage type rape (Brassica napus)

<400> 34

ggaaatacaa cattgtcagt atattagata tctattcgag cacatctcta gactatataa 60

agaaagaaaa atatatagag agagagagga gagagatggg gaggcaacca tgttgtgaca 120

aagtaggctt gaagaaaggg ccatggactg ttgaagaaga caagaagctc atcaacttca 180

tcctcaccaa tggccaatgc tgttggagag ctcttcccaa gctttctggt cgattttctc 240

tctttatttc ctccttaaac gacatctttt gggatttctc atgtgtaaaa gttcactttc 300

ttgcttcttg tgtgtttagc ctttctctac accaactgat gctatttttt gggtgctatt 360

tctctacacc gacgaaactg atgctcttat ttaggatctc gtattttaaa agttcacttc 420

cttaaaacgt ccaattttat tactagtaat ttatatttct ctactttctt cttgtcagca 480

gtttttgtag taatattttc tctacttttg tgtaaattaa tttgcatgag ttattttggt 540

atatgcatgc tagtatagat gttcaccaac aatcaaatat acctttataa attattttca 600

agaaacaatt ttgatgcaga agaatcatat gaattcttaa ctttcctttt tgaaatagaa 660

tatttctcta tcggttggac ctaaaaacaa aagtctcaaa aaaacatagt gcttgatatt 720

cgtggtcctc acacgttatt atttgatctt gatcaatttt tttgttttgt ttttggcttt 780

ttgtttagtg cttcctgcct agtcaaaata tatttcgtat ggttttaact ttcaatcagt 840

ccaattgtta atttaattac tttggttaat taactaaatg caggactatt gaggtgtggt 900

aaaagctgca gattaagatg gataaattat ttgagacctg atttaaaaag aggtctttta 960

tctgaaaatg aagagcagat ggtcattgat cttcatgccc aacttggcaa taggtttgct 1020

ctctttttct tcaaaataat tgttatttca tattataaac ataatttact tacgttaact 1080

tgacattgta actaactaat aatataataa aatatttaaa gattatggcc atttgatttc 1140

aatattatta agggtattgg agaaacacaa aagtcattat atgacgcaaa ttaactttat 1200

cacttttttg catgaaagag attggctaat ccacatcttt atttactcaa attttctttt 1260

taaccccagt agtttaatta agtcggtgtg atgaaaatgt ttgaacagtc gaaatcatta 1320

gttaaattca cacttataaa attttcaaat attttatttt tatttttcgt gcagatggtc 1380

aaagattgcc tctcatctac caggaagaac tgataatgaa attaagaacc attggaacac 1440

acacataaag aaaaaactaa ggaagatggg tattgaccct atgacccata agcctctctc 1500

tgaacaagac ggtgcgcaac aacaagctca ggggataaag aaaagcttag tgtctcgtga 1560

tgagaagaac acaaaccaag gccaagaaga tcaacaacaa actagaaaag aagaagatca 1620

acaacatcaa ctagggaaag atttggagaa gaacaacaca cccatttcta ctgatgatgg 1680

gttttgcatt gatgatatcc cattactcag tccacatgag atatttgtgg acatctcttc 1740

tgctcaccat catactagtg atgataatgc caacattaac attagtaact ccacttcacc 1800

ttcttcgtct tcctcttcta cttcgtcatg catatcatca ctaccgggtg atgagttctc 1860

aaagtttctt gatgaaatgg agattgttga tctcaagtgg ctctcatccg atgattcatc 1920

agggggtgac attaattaca acgacggcaa gttcaacaac aatgttgata cgatgagctt 1980

gtgggacatc aatgatttga gcagcttgga attgttttat gaatgatcat gatgatagtg 2040

tggttgaagc ggatgttcaa gaatggtttt agatcatgat tcatggacat ttgatcttct 2100

ttaactttta gttttttttt tcttctcttg ttgtttttaa ttgtttttgt ttgtttgttt 2160

gggtttggcc ttggtgttgc agtttagtgg atgattaggt gtaaaagtac atatgtaatt 2220

aaagattgca attgtgaaca tacctttcac tg 2252

<210> 35

<211> 2237

<212> DNA

<213>cabbage type rape (Brassica napus)

<400> 35

cttgtttgag catatctcta gactaatata tatatatata tatatataga gagagagaga 60

cagagggtgc gaggagtgaa gatggggagg cagccatgtt gtgacaaagt agggctgaag 120

aaagggccat ggacagctga ggaagacaag aagctcatca acttcatcct caccaatggc 180

cactgctgtt ggagagctct tcccaagctt tctggtatat tttctttctt tcttttctcc 240

acatgcgacg tccttgggat ttctcatgtg taaaaagttc gtttttttct taacacatgt 300

gtttttacat tttctcctac caaaaaaacg atatgctatt tttgatatat ggattatcgt 360

atcttaaaag tgaactttct taaaacgtcc ccttttattc ctactgattt ctattttatc 420

tattttcttg tttttgctga cactaataat aatacttctc tacccttttt taattaattt 480

gcatgagttc ttttgctata tctatactag tatagttttg caccaatcaa atgaatttac 540

tattttcagt aatagtttca atgcaggaga ataatattca ttgttaccgt cctttttgaa 600

atggaataat tctctatctt ttcgacttac gaaatccgat caatcatggt gtttggcccg 660

gacaaaaatc aatgtttgat actggtggtc ctacacgtta ttatttgttc ttgataattt 720

tttgttctgt tttgttttca cttgttcctt cttaatcagt ttatctctcg tatatttaac 780

tagtttccaa tcattctgat cgttaatcta gttactttgg ttaatcaatt aaatcaggac 840

tcttgaggtg tggaaaaagc tgcagactaa gatggatcaa ttatttgaga cctgatttaa 900

aaagaggtct tttatcagaa tgtgaagaac agatggtcat tgatcttcat gcccaacttg 960

gcaataggtt tattctcttt ctcttgaaaa tcattgttat tccatatttt aaaaaatcct 1020

caaatttata aacagtaatt ttacctacct tttaaaaaaa aaactaattt tacctgcctt 1080

aactttgatg ttgtaactaa ctcgtaataa aaatataaaa atattgtggc cattagattt 1140

caatattatt aaggtcacgg aggaacacaa aagtcacaat agaacactga acacgcaaac 1200

tttactattt ttttctgtat gaaagagatt ggccaatcta catctcatat ttaaatttaa 1260

tcgtgcgttt ttaaccacgt acggtagttt aatttagtaa gtctgatgat aatgactgaa 1320

cagtcgaaat catgagttaa ttaaacttac ttaagtatcg aatcatttta atttttgtgc 1380

agatggtcaa agattgcctc tcatctacca ggaagaactg ataatgaaat aaagaaccat 1440

tggaacacac acataaagaa aaaactaagg aagatgggca ttgaccctat gacccataag 1500

cctctctctg aacaagacgg gtcacagcaa gctcaaggga gcaagagttt agtgcctcat 1560

gatgacaaga acaaaaaaca agaccaagaa gataaacaaa ctaaagaaaa acaagaacac 1620

catcaactag ggatagattt ggacaagagc aacaaaacaa tatctactga tgatgggttt 1680

tgcattgatg aagtcccgtt gctcgatcca catgagatat tactggacat ctcttttgct 1740

gatcatcata ctagtgatga aaatgctcac attaacatta gtaactccac ttctccttct 1800

tcctcttcta cctcgtcgtg tatatcatca gtaccgggtg aggagttctc gaagtttctt 1860

gatgaaatgg acactattga cctcaagtgg ctctcatccg atcattcatc agaggataat 1920

attatttaca aagacgacaa gttccacaac aatgttgata cgatgaactt gtgggacata 1980

aattatttga gcagcttgga attgtttatg aatgatcatg atgatggttt tgttggaagc 2040

ggatgttcaa gaatggttta gatcaatata catgaccatt ttactttcag tttttttttc 2100

atgttgtttt taattagttc tttttgtttg ttttcgaagt gggcgatttg gatttggcct 2160

tggtgttgca atttagtgga tgattaggtg taaaatacat atgtaataaa tattgcaatt 2220

gtgaacacac ttttctc 2237

<210> 36

<211> 2256

<212> DNA

<213>cabbage type rape (Brassica napus)

<400> 36

atatacatta taacttgtaa gtatattagg tacttgtttg agcatatctc tagactaata 60

gatatatata tatatagaga gagagagcca gagggtgcga ggagtgaaga tggggaggca 120

gccatgttgt gacaaagtag ggctgaagaa agggccatgg acagctgagg aagacaagaa 180

gctcatcaac ttcatcctca ccaatggcca ctgctgttgg agagctcttc ccaagctttc 240

tggtatatct tctctctttc tttcttttct ccatatgcga cgtccttggg atttctcatg 300

tgtaaaaagt ttttttttct taacacatgt gtttttacat tttctcttac caaaaaaatg 360

atatgctatt tttgatatat ggattatcgt attttaaaag ttaactttct taaaacgtcc 420

ccttttattc ctactgattt ctattttatc tattttcttg tttttgctga cactgataat 480

aatacttctc tacctttttt aattaatctg catgagttct tttgctatat ctatactagt 540

atagttttgc accaaatcaa atgaatttac tatttttcag taataggttc aatgcaggag 600

aatcatattc aatgataccg ccccttttga aatggaagaa ttctatatct tttctactgt 660

aaaaacctat cgaccatgat gattggcctt cgactaaaga gtggcctcta ctgccgccca 720

tttatatagt ttatgttcat gaaaaattat gtgttctgag acgtctgggt cacttttcag 780

ggaaaagtcc gcgtaacccc tatttgttca tttttctctt tccaatcatt ctgatcgtta 840

atctaattac tttggttaat caattaaatc aggactcttg aggtgtggaa aaagctgcag 900

actaagatgg atcaattatt tgagacctga tttaaaaaga ggtcttttat cagaatgtga 960

agaacagatg gtcattgatc ttcatgccca acttggcaat aggtttattc tctttctctg 1020

gaaaatcatt gttattccat atttttaaaa atcctcaaat ttataaacag taattttacc 1080

taccttttta aaaaaaacta atttcacctg ccaaactttt atgttgtaac taactcgtaa 1140

taaaaatata aaaaatattg tggccattag acttcaatat tattaaggtc tcggaggaac 1200

acaaaagtca caatagaaca ctgaacacgc aaactttact atttcttctg tatgaaagag 1260

attggccaat ctacatctca tatttaaatt taatcgtgcg tttttaacca cgtacggtag 1320

tttaatttag taggtctgat gataatgact gaacagtcga aatcatgagt taattaaact 1380

taagtttcga atcattttaa tttttgtgca gatggtcaaa gattgcctct catctaccag 1440

gaagaactga taatgaaata aagaaccatt ggaacacaca cataaagaaa aaactaagga 1500

agatgggcat tgaccctatg acccataagc ctctccctga acaagacggt tcacagcaag 1560

ctcaagggag caagagttta gtgcctcatg atgacaagaa caaaaaacaa gaccaagaag 1620

ataaacaaac taaagaaaaa caagaacacc atcaactagg gatagatttg gacaagagca 1680

acaaaacaat atctactgat gatgggtttt gcattgatga agtcccgttg ctcgatccac 1740

atgagatatt actggacatc tcttttgctc agcatcatac tagtgatgaa aatgctcaca 1800

ttaacattag taactccact tctccttctt cctcttctac ctcgtcgtgt atatcatcag 1860

taccgggtga ggagttctcg aagtttcttg atgaaatgga gactattgac ctcaagtggc 1920

tctcatccga tcattcatca ggggataata ttatttacaa agacgacaag ttccacaaca 1980

atgttgatac gatgaacttg tgggacataa attatttgag cagcttggaa ttgtttatga 2040

atgatcatga tgatggtttt gttggaagcg gatgttcaag aatggtttag atcaatattc 2100

atgaccattt tactttcagt tttttttttc atgttgtttt taattagttc tttttgtttg 2160

ttttcgaagt gggcgattcg gatttggccg tggtgttgca atttagtgga tgattaggtg 2220

taaaatacat atgtaataaa gattgcaatt gtgaac 2256

<210> 37

<211> 2295

<212> DNA

<213>Chinese cabbage (Brassica rapa)

<400> 37

ggaaatacaa cattgtcagt atattagata tctatttgag cacatctcta gactatataa 60

agtaagaaaa atatatatag agagaggaga gagatgggga ggcaaccatg ttgtgacaaa 120

gtaggcttga agaaaggccc atggactgtt gaagaagaca agaagcttat caacttcatc 180

ctcaccaatg gccaatgctg ttggagagct cttcccaagc tttctggtcg attttctctc 240

tttatttcct ccttaaacga catcttttgg gatttctcat gtgtaaaagt tcactttctt 300

gcttcttgtg tgtttagcct ttctctacgc caactgacgc ttttttggtg ctatttctct 360

acaccgacga aactgatgct attttgattt ttagatactc gtcttttaaa agttcacttc 420

cttaaaacgt ccaattttat tactagtaat ttctatttct ctactttctt cttgtcagca 480

gtttttatag taatattttc tttacttttg tgtaaattaa tttgcatgag ttattttggt 540

atatgcatgc tagtatagat gttcaccaac aatcaaatat acctttagaa attagtttca 600

agaaacaatt ttgatgcagg agaatcatat gaattcttaa ctttcctttt tgaaatacaa 660

tatatatttt aatatcggtt ggacctaaaa acaaaagtct caaaacatat aattagacta 720

ttaatcatag gctcatagcg tctggcccga aaagaaatat agtgcttgat attcgtggtc 780

ctcacacgtt attagttgat cttgataaaa aaaaattgtt ttgtttttgg ctttttgttt 840

agtgcttcct gccaaaatat atttcgtatg gttttaactt tcaatcaatc tgattgttaa 900

tttaattact ttggttaatt aactaaatgc aggactattg aggtgtggta aaagctgcag 960

attaagatgg ataaattatt tgagacctga tttaaaaaga ggtcttttat cagaaaatga 1020

agagcagatg gtcattgatc ttcatgccca acttggcaat aggtttatcc tctttttatt 1080

caaaataatt gttatttcat attataaaca taatttactt accttatttg acattgtaac 1140

taactcataa tataatgaaa tgtttaaaga ttatggacat tagatttcaa tattaaaggt 1200

attggagaag cacaaaagtc attatatgac gcaaacttta tcactttttt tgcatgaaag 1260

agattggcta atccacattt ttaattactc atattttctt tcaaacccaa tatagtttaa 1320

ttaagtcggt gtgatgaaaa tgtttgaaca gtcgaaatca ttagtgaaat tatcacttat 1380

aaaattttca aatattttat ttttattttt gtgcagatgg tcaaagattg cctctcatct 1440

accaggaaga actgataatg aaattaagaa ccattggaac acacacataa agaaaaaact 1500

aaggaagatg ggtattgacc ctatgaccca taagcctctc tctgaacaag acggtgcgca 1560

acaacaagct caggggagaa agaaaagctt agtgtctcgt gatgagaaga acacaaacca 1620

aggccaagaa gatcaacaac aaactagaaa agaagaagat caacaacatc aactagggaa 1680

agatttggag aagaacaaca cacccatttc tactgatgat gggttttgca ttgatgatat 1740

cccattactc agtccacatg agatatttgt ggacatctct tctgctcacc atcatactag 1800

tgatgataat gccaacatta acattagtaa ctccacttca ccttcttcgt cttcctcttc 1860

tatctcgtcg tgcatatcat cactaccggg tgatgagttc tcaaagtttc ttgatgaaat 1920

ggagattgtt gacctcaagt ggctctcatc cgatgattca tcagggggta acattaatta 1980

caacgacggc aagttcaaca acaatgttga tacgatgagc ttgtgggaca tcaatgattt 2040

gagcagcttg gaattgttta tgaatgatca tgatgatagt gtggttgaag cggatgttca 2100

agaatggttt tagatcatga ttcatggaca tttgatcttc tttaactttt agtttttttt 2160

tcttctcatg ttgtttttaa ttgtttttgt ttgtttgttt gggtttggcc ttggtggtgg 2220

atgattaggt gtaaaagtac atatgtaatt aaagattgca attgtgaaca tacttttctc 2280

tgtttaacct cgtat 2295

<210> 38

<211> 2240

<212> DNA

<213>Chinese cabbage (Brassica rapa)

<400> 38

cttgtttgag catatctcta gactaatata tatatatata tatatatata tagagagaga 60

gacagagggt gcgaggagtg aagatgggga ggcagccatg ttgtgacaaa gtagggctga 120

agaaagggcc atggacagct gaggaagaca agaagctcat caacttcatc ctcaccaatg 180

gccactgctg ttggagagct cttcccaagc tttctggtat attttctttc tttcttttct 240

ccacatgcga cgtccttggg atttctcatg tgtaaaaagt tcgttttttt cttaacacat 300

gtgtttttac attttctcct accaaaaaaa cgatatgcta tttttgatat atggattatc 360

gtatcttaaa agtgaacttt cttaaaacgt ccccttttat tcctactgat ttctatttta 420

tctattttct tgtttttgct gacactaata ataatacttc tctacccttt tttaattaat 480

ttgcatgagt tcttttgcta tatctatact agtatagttt tgcaccaatc aaatgaattt 540

actattttca gtaatagttt caatgcagga gaataatatt cattgttacc gtcctttttg 600

aaatggaata attctctatc ttttcgactt acgaaatccg atcaatcatg gtgtttggcc 660

cggacaaaaa tcaatgtttg atactggtgg tcctacacgt tattatttgt tcttgataat 720

tttttgttct gttttgtttt cacttgttcc ttcttaatca gtttatctct cgtatattta 780

actagtttcc aatcattctg atcgttaatc tagttacttt ggttaatcaa ttaaatcagg 840

actcctgagg tgcggaaaaa gctgcagact aagatggatc aattatttga gacctgattt 900

aaaaagaggt cttttatcag attgtgaaga acagatggtc attgatcttc atgcccaact 960

tggcaatagg tttattctct ttctcttgaa aatcattgtt attccatatt ttaaaaaatc 1020

ctcaaattta taaacagtaa ttttacctac ctttttaaaa aaaaactaat tttacctgcc 1080

ttaactttga tgttgtaact aactcgtaat aaaaatataa aaatattgtg gccattagat 1140

ttcaatatta ttaaggtcac ggaggaacac aaaagtcaca atagaacact gaacacgcaa 1200

actttactat ttttttctgt atgaaagaga ttggccaatc tacatctcat atttaaattt 1260

aatcgtgcgt ttttaaccac gtacggtagt ttaatttagt aagtctgatg ataatgactg 1320

aacagtcgaa atcatgagtt aattaaactt acttaagtat cgaatcattt taatttttgt 1380

gcagatggtc aaagattgcc tctcatctac caggaagaac tgataatgaa ataaagaacc 1440

attggaacac acacataaag aaaaaactaa ggaagatggg cattgaccct atgacccata 1500

agcctctccc tgaacaagac gggtcacagc aagctcaagg gagcaagagt ttagtgcctc 1560

atgatgacaa gaacaaaaaa caagaccaag aagataaaca aactaaagaa aaacaagaac 1620

accatcaact agggatagat ttggacaaga gcaacaaaac aatatctact gatgatgggt 1680

tttgcattga tgaagtcccg ttgctcgatc cacatgagat attactggac atctcttttg 1740

ctgatcatca tactagtgat gaaaatgctc acattaacat tagtaactcc acttctcctt 1800

cttcctcttc tacctcgtcg tgtatatcat cagtaccggg tgaggagttc tcgaagtttc 1860

ttgatgaaat ggacactatt gacctcaagt ggctctcatc cgatcattca tcagaggata 1920

atattattta caaagacgac aagttccaca acaatgttga tacgatgaac ttgtgggaca 1980

taaattattt gagcagcttg gaattgttta tgaatgatca tgatgatggt tttgttggaa 2040

gcggatgttc aagaatggtt tagatcaata tacatgacca ttttactttc agtttttttt 2100

ttcatgttgt ttttaattag ttctttttgt ttgttttcga agtgggcgat ttggatttgg 2160

ccttggtgtt gcaatttagt ggatgattag gtgtaaaata catatgtaat aaatattgca 2220

attgtgaaca tacttttctc 2240

<210> 39

<211> 2259

<212> DNA

<213>wild cabbage (Brassica oleracea)

<400> 39

ggaaatacaa cattgtcagt atattagata tatttgagca catctctaga ctatataaag 60

taagaaaaat atatatagag agaggagaga gatggggagg cagccatgtt gtgacaaagt 120

aggcttgaag aaagggccat ggactgttga agaagacaag aagctcatca acttcatcct 180

caccaatggc caatgctgtt ggagagctct tcccaaactt tctggtcgat tttctctctt 240

tatttcctcc ttaaacgaca tcttttggga tttctcatgt gtaaaagttc actttcttgc 300

ttcttgtgtg tttagccttt ctctacacca actgatgcta ttttttgggt gctatttctc 360

tacaccgacg aaactgatgc tcttatttag gatctcgtat tttaaaagtt tcacttcctt 420

aaaacgtcca attttattac tagtaattta tatttctcta ctttcttctt gtcagcagtt 480

tttatagtaa tattttctct acttttgtgt aaattaattt gcattagtta ttttggtata 540

tgcatgctag tatagatgtt caccaacaat caaatatacc tttagaaatt attttcaaga 600

aacaattttg atgcagaaga atcatatgaa ttcttaactt tcctttttaa aatagaatat 660

ttctctatcg gttggaccta aaaacaaaag tctcaaaaaa acatagtgct tgatattcgt 720

ggtcctcaca cgttattatt tgatcttgat caattttttt gttttgtttt tggctttttg 780

tttagtgctt ccttcctagt caaaatatat ttcgtatggt tttaactttc aatcagtcca 840

attgttaatg taattacttt ggttaattaa ctaaatgcag gactattgag gtgtggtaaa 900

agctgcagat taagatggat aaattatttg agacctgatt taaaaagagg tcttttatca 960

gaaaatgaag agcagatgat cattgatctt catgcccaac ttggcaatag gtttgctctc 1020

tttttcttca aaataattgt tatttcatat tataaacata atttacttac gttaacttga 1080

cattgtaact aactaataat ataataaaat atttaaagat tatggccatt tgatttcaat 1140

attattaagg gtattggaga aacacaaaag tcattatatg acgcaaatta actttatcac 1200

ttttttgcat gaaagagatt ggctaatcca catctttatt tactcatatt ttctttttaa 1260

ccccagtact ttaattaagt cggtgtgatg aaaatgtttg aacagtcgaa atcattagtt 1320

aaaatcacac ttataaaatt ttcaaatatt ttatttttat tttttgtgca gatggtcaaa 1380

gattgcctct catctaccag gaagaactga taatgaaatt aagaaccact ggaacacaca 1440

cataaagaaa aaactaagga agatgggtat tgaccctatg acccataagc ctctctctga 1500

acaagaaggt gcgcagcaac aagctcaggg gacaaagaaa agcttagtgt ctcgtgatga 1560

gaagaaaaga aaccaaggcc aagaagatca acaaactaga aaagaagaag atcaacaaca 1620

tcaactaggg aaacatttgg acaagaacaa cacacccatt tctactgatg atgggttttg 1680

cattgatgat atcccattac tcaatccaca tgagatattt gtggacatct cttctgctca 1740

ccatcatact agtgatggta atgccaacat taacattagt aactccactt caccatcttc 1800

gtcttcctct cctacctcgt cgtgcatatc atcactacca ggtgatgagt tctcaaagtt 1860

tcttgatgaa atggagattg ttgacctcaa gtggctctca tccgatgatt cgtcaggggg 1920

tgacattaat tacaaagacg gaaagttcaa taacaatgtt gatacgatga acttgtggga 1980

catcaatgat ttgagcagct tggaattgtt tatgaatgat cacgatgata gtgtggttgg 2040

aagcggatgt tcaagaatgg ttttagatca tgattcatgg acatttgatc ttctttaact 2100

tttagttttt ttttcttctc ttgttgtttt taattgtttt ttgtttgttt gtttgggttt 2160

ggcctcggtg ttgcagttta gtggatgatt aggtgtaaaa gtacatatgt aattaaagat 2220

tacaattgtg aacatacttt tctctgttta ttttgacat 2259

<210> 40

<211> 2258

<212> DNA

<213>wild cabbage (Brassica oleracea)

<400> 40

atatacatta taacttgtaa gtatattagg tacttgtttg agcatatctc tagactaata 60

gatatatata tagagagaga gagagccaga gggtgcgagg agtgaagatg gggaggcagc 120

catgttgtga caaagtaggg ctgaagaaag ggccatggac agctgaggaa gacaagaagc 180

tcatcaactt catcctcacc aatggccact gctgttggag agctcttccc aagctttctg 240

gtatatcttc tctctttctt tcttttctcc atatgcgacg tccttgggat ttctcatgtg 300

taaaaagttt ttttttctta acacacgtgt ttttacattt tctcttacca aaaaaacgat 360

atgctatttt tgatatatgg attatcgtat tttaaaagtt aactttctta aaacgtcccc 420

ttttattcct actgatttct attttatcta ttttcttgtt tttgctgaca ctgataataa 480

tacttctcta ccttttttaa ttaatctgca tgagttcttt tgctatatct atactagtat 540

agttttgcac caatcaaatg aatttactat tttcagtaat agtttcaatg caggagaatc 600

atattcattg ttaccgtcct ttttgaaatg gaataattct ctatcttttc gacttacaaa 660

atccgatcaa tcatggtgtt tggcccggac aaaaatcagt gtttgatact ggtggtccta 720

cacgttatta tttgttcttg ataatttttt gttctgtttt gttttgtttt cacttgttcc 780

ttcttattca gtttatctct cgtatattta actagtttcc aatcattctg atcgttaatc 840

taattacttt ggttaatcaa ttaaatcagg actcttgagg tgtggaaaaa gctgcagact 900

aagatggatc aattatttga gacctgattt aaaaagaggt cttttatcag aatgtgaaga 960

acagatggtc attgatcttc atgcccaact tggcaatagg tttattctct ttctctggaa 1020

aatcattgtt attccatatt tttaaaaatc ctcaaattta taaacagtaa ttttacctac 1080

cttttaaaaa aaactaattt tacctgccaa actttgatgt tgtaactaac tcgtaataaa 1140

aatataaaaa atattgtggc cattagactt caatattatt aaggtctcgg aggaacacaa 1200

aagtcacaat agaacactga acacgcaaac tttactattt cttctgtatg aaagagattg 1260

gccaatctac atctcatatt taaatttaat cgtgcgtttt taaccacgta cggtatagtt 1320

taatttagta ggtctgatga taatgactga acagtcgaaa tcatgagtta attaaactta 1380

cttaagtttc gaatcatttt aatttttgtg cagatggtca aagattgcct ctcatctacc 1440

aggaagaact gataatgaaa taaagaacca ttggaacaca cacataaaga aaaaactaag 1500

gaagatgggc attgacccta tgacccataa gcctctccct gaacaagacg gttcacagca 1560

agctcaaggg agcaagggtt tagtgcctca tgatgacaag aacaaaaaac aagaccaaga 1620

agataaacaa actaaagaaa aacaagaaca ccatcaacta gggatagatt tggacaagag 1680

caacaaaaca atatctactg atgatgggtt ttgcattgat gaagtcccgt tgctcgatcc 1740

acatgagata ttactggaca tctcttttgc tcagcatcat actagtgatg aaaatgctca 1800

cattaacatt agtaactccg cttctccttc ttcctcttct acctcgtcgt gtatatcatc 1860

agtaccgggt gaggagttct cgaagtttct tgatgaaatg gagactattg acctcaagtg 1920

gctctcatcc gatcattcat caggggataa tattatttac aaagacgaca agttccacaa 1980

caatgttgat acgatgaact tgtgggacat aaattatttg agcagcttgg aattgtttat 2040

gaatgatcat gatgatggtt ttgttggaag cggatgttca agaatggttt agatcaatat 2100

tcatgaccat tttactttca gttttttttt tcatgttgtt tttaattagt tctttttgtt 2160

tgttttcgaa gtgggcgatt tggatttggc cgtggtgttg caatttagtg gatgattagg 2220

tgtaaaatac atatgtaata aagattgcaa ttgtgaac 2258

Claims (8)

1. striking application of the expression for dropping cabbage type rape and its parent species MYB43 in raising disease resistance of plant, feature exists In: the full length cDNA sequence of cabbage type rape MYB43 family is SEQ ID NO.25, SEQ ID NO.26, SEQ ID Shown in NO.27 and SEQ ID NO.28, the full length cDNA sequence of parent species Chinese cabbage MYB43 family is SEQ ID NO.29, SEQ Shown in ID NO.30, the full length cDNA sequence of parent species wild cabbage MYB43 family is SEQ ID NO.31, SEQ ID NO.32 institute Show.

2. application according to claim 1, it is characterised in that: the overall length gDNA sequence of cabbage type rape MYB43 family It is classified as SEQ ID NO.33, SEQ ID NO.34, shown in SEQ ID NO.35 and SEQ ID NO.36, parent species Chinese cabbage The overall length gDNA sequence of MYB43 family is parent species wild cabbage MYB43 family shown in SEQ ID NO.37, SEQ ID NO.38 Overall length gDNA sequence be SEQ ID NO.39, SEQ ID NO.40 shown in.

3. application according to claim 1, it is characterised in that: the method for striking drop MYB43 is, for cabbage type rape, white Dish, wild cabbage MYB43 gene family construct rna interference vector, and the transformant of acquisition is then converted cabbage type rape or its parent Species obtain the plant for striking drop MYB43.

4. application according to claim 3, it is characterised in that: the rna interference vector is as shown in SEQ ID NO.25 604~1084bp shown in 602-1081bp, SEQ ID NO.26,578~1058bp, SEQ ID shown in SEQ ID NO.27 606~1086bp shown in No.28,580 shown in 602~1081bp, SEQ ID No.30 shown in SEQ ID No.29~ The antisense fragments of 597~1077bp shown in 1060bp, SEQ ID No.31,604~1084bp shown in SEQ ID No.32 It is inserted between the CaMV35S promoter and BnPAP2 Intron II of pFGC5941M carrier and obtains；

Or 602-1081bp shown in SEQ ID NO.25,604~1084bp shown in SEQ ID NO.26, SEQ ID NO.27 Shown in 602 shown in 606~1086bp, SEQ ID No.29 shown in 578~1058bp, SEQ ID No.28~ 580~1060bp shown in 1081bp, SEQ ID No.30,597~1077bp, SEQ ID shown in SEQ ID No.31 The sense fragment of 604~1084bp shown in No.32 is inserted into pFGC5941M between BnPAP2 Intron II and OCS3 ', Form RNAi carrier pFGC5941M-BMYB43I.

5. application according to claim 3, it is characterised in that: the transformant is the crown gall containing the rna interference vector Agrobacterium tumefaciens attachment.

6. the described in any item applications of Claims 1 to 5, it is characterised in that: shown plant is cabbage type rape, Chinese cabbage or sweet It is blue.

7. the expression for striking drop cabbage type rape and its parent species MYB43 is reducing cabbage type rape or its parent species stem core Application in cup fungi scab length, it is characterised in that: the full length cDNA sequence of cabbage type rape MYB43 family is SEQ ID NO.25, SEQ ID NO.26, shown in SEQ ID NO.27 and SEQ ID NO.28, the overall length of parent species Chinese cabbage MYB43 family CDNA sequence is shown in SEQ ID NO.29, SEQ ID NO.30, and the full length cDNA sequence of parent species wild cabbage MYB43 family is Shown in SEQ ID NO.31, SEQ ID NO.32.

8. the expression for striking drop cabbage type rape and its parent species MYB43 is improving cabbage type rape or its parent species unit plane Application in product yield, it is characterised in that: the full length cDNA sequence of cabbage type rape MYB43 family is SEQ ID NO.25, SEQ ID NO.26, shown in SEQ ID NO.27 and SEQ ID NO.28, the overall length of parent species Chinese cabbage MYB43 family CDNA sequence is shown in SEQ ID NO.29, SEQ ID NO.30, and the full length cDNA sequence of parent species wild cabbage MYB43 family is Shown in SEQ ID NO.31, SEQ ID NO.32.