CN105063085B - The application of cabbage type rape gene BnMPK3 and its anti-sclerotinia sclerotiorum - Google Patents

Abstract

The invention discloses a kind of genes of control cabbage type rape sclerotinioseBnMPK3And application, belong to plant genetic engineering field；Relate generally to a kind of gene of control cabbage type rape sclerotinioseBnMPK3Separation clone, functional verification and application,BnMPK3Gene has the function of control sclerotium disease, and the present invention is by experimental verification when sclerotiniose handles cabbage type rapeBnMPK3Expression quantity significantly improve, improved by technique for gene engineeringBnMPK3The expression quantity of gene can control sclerotium disease, to improve the disease resistance and yield of cabbage type rape and other crops.

Description

Cabbage type rape geneBnMPK3And its application of anti-sclerotinia sclerotiorum

Technical field

The present invention relates to a kind of genes of control cabbage type rape sclerotinioseBnMPK3And its or its functional analogue regulation and control Application of the Genes For Plant Tolerance sclerotiniose in agricultural production, belongs to plant genetic engineering field.

Background technology

Rape occupies an important position as a kind of important oil crops in the oil seed production of China.China's rape 7,000,000 hectares of annual planting area（100000000 mu）, global 1/3 is accounted for, is occupied first place in the world, produced rapeseed oil accounts for China and entirely eats 40% supplied with vegetable oil（Shen Jinxiong etc., china rape production and genetic improvement pot ential and brassinosteroid biosynthesis diesel oil development prospect [J], Hua Zhong Agriculture University's journal, 2007,894-899 pages of the phase of volume 26 6）.In addition, rapeseed oil is also the biology for being suitable for production Diesel oil, rape is by European and American countries as the important source material crop for solving global energy shortage.Therefore rape is not only edible oil Main source, and the important source material crop of global energy shortage is solved, with highly important strategic position.

However, the vegetable seed output in China is only capable of meeting the consumption demand of the country 2/3 at present, import is largely relied on.China More than 600 ten thousand tons of imported plants oil in 2005 is maximum oil plant importer in the world, as the growth of the size of population and the people are given birth to The flat raising of running water, by current production scale, notch will become much larger（Wang Hanzhong, the present situation of Chinese oil plant industry development, Problem and countermeasure [J], Chinese oil crops journal, 2005,100-105 pages of the phase of volume 27 4）.It is considerably long at present and in the future Time in, China will all face the severe situation of edible oil and fossil energy shortage.

Sclerotinia sclerotiorum（Sclerotinia sclerotiorum（Lib.）de Bary）It is limitation China's Rape-seed production One of Main Factors.In the middle and lower reach of Yangtze River and southeastern coast rape main producing region, the incidence of sclerotinia sclerotiorum is very high, production loss Up to 50% or more；For national occurring area at 70,000,000 mu or more, production loss is up to 30%, and 50-80% is reached when serious.At present The control method of sclerotinia sclerotiorum is mainly the selection and breeding of Techniques For Chemical Control and disease-resistant variety, but chemical prevention there are preventive effects not High, cause of disease is difficult to eradicate, florescence spray is difficult and the problems such as pesticide residue, environmental pollution；Conventional breeding is looked for because being difficult to The shortcomings of to effective resistant material and very slow speed, it is also difficult to effectively solve the problems, such as disease-resistant.Sclerotiniose is fought as a result, Rape variety carries out molecular improvement, and the anti-sclerotiniose kind of quickly breeding is the Critical policies for improving vegetable seed yield per unit area.

Gene engineering method, by genetic manipulation, is improved on a molecular scale by pre-designed target and plan The technology of kind.Institute of Crop Science, Chinese Academy of Agricultural Science is found that a plant disease resistance genes are gene a or gene b, It will be significantly increased to the resistance of plant after gene a or gene b modifications（Application number：201110048518.9 2011）.And as oil Dish sclerotiniose is this kind of is difficult to find in host plant kind the disease of antigen, and gene engineering method provides a kind of solve the problems, such as Approach.

Mitogen-activated protein kinase（Mitogen-activited protein kinases, MAPKs）It is a kind of silk Propylhomoserin/Serineprotein kinase carries out response in cell to various growth factors, rapidly in its serine, threonine residues Site phosphorylation and be activated.It is widely present in various eucaryotes, and MAPK grades are formed with some other signaling molecules Connection approach（MAPK cascades）, the approach and hormone response, cell differentiation and growth course are closely related.Shandong agricultural is big It learns the study found that cotton mitogen activated protein kinase geneGhMAPK6Can make cotton leaf area increase, fruit pod number and Setting percentage improves, to improve output of cotton（Guo Xingqi etc., 2010；Application number：201010590833）.In plant, MAPK In addition to having outside the Pass with many vital movements, coping with, hot and cold, oxidation, ultraviolet light, arid, pathogen invasion etc. are a variety of abiotic It plays an important role in terms of biotic.There is scholar's discovery, riceOsMPK4/OsMPK5It is related with rice low-temperature resistance （Huang Fu et a1. Expression ofOryzasativa MAP kinase gene is developmentally Regulated and stress-responsive [J] .Physiol Plant, 2012,114 phases, 572-580 pages）.Central China Agriculture university's discovery, trifoliate orange mitogen-activated protein kinase genePtMAPKTransgenic tobacco plant drought-resistant ability can be improved, New genetic resources are provided for Genes For Plant Tolerance abiotic stress Molecular design breeding（Liu Jihong etc., 2010；Application number： 201010596044）.Mitogen-activated protein kinase geneMPK3It is important a member of MAPK families, the research table of Mao etc. It is bright, in arabidopsisMPK3Activation can be passed throughWRKY33The synthesis for promoting plant protective plant protecting agent, to play anti-arabidopsis gray mold Effect（Mao et a1. Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis[J] .Plant Cell, 2011,23 phases, page 1639-1653).It is proved gene at presentBnWRKY33There is anti-sclerotinia sclerotiorum Effect（Wang Zheng et a1. Overexpression ofBnWRKY33 in Oilseed Rape Enhances Resistance to Sclerotinia sclerotiorum. Molecular Plant Pathology[J] .Molecular Plant Pathology, 2014）.Therefore it is presumed that,BnMPK3Gene, which also has, improves Wild cabbage type oil Dish is to the function of resistance to sclerotinia sclerotiorum, and in canola, existing researcher clonesBnMPK3Gene,（Liang et al. Identification and analysis of MKK and MPK gene families in canola (Brassica NapusL.) .BMC Genomics, 2013 years）, but at present forBnMPK3The research of anti-sclerotiniose this aspect yet there are no The report of document and patent.The study found that after sclerotiniose is to rape Stress treatment,BnMPK3Expression significantly improve, in turn, Cabbage type rapeBnMPK3Transgenosis is overexpressed plant and significantly increases resistance to sclerotinia sclerotiorum, this result is anti-in enhancing sclerotinia sclerotiorum Property and improve vegetable seed oil yield on it is with important application prospects.

Invention content

It is an object of the invention to overcome defect existing in the prior art, according to a kind of control crop from rape The gene of sclerotiniose, is named asBnMPK3, nucleotides sequence is classified as shown in SEQ ID NO.1；BnMPK3The egg of gene code White matter, amino acid sequence are shown in SEQ ID NO.2.

Present invention offer carriesBnMPK3The recombinant vector 1300-35S-MPK3-NOS of gene.

The specific preparation method of the recombinant vector 1300-35S-MPK3-NOS is：In carrier pCAMBIA1300（It is purchased from Beijing prosperity biotech firm of ancient cooking vessel state）Upstream EcoRI/KpnI restriction enzyme sites at be added CaMV 35S strong promoters, downstream CaMV Nos terminators are added at BamHI/HindIII restriction enzyme sites, are transformed into carrier pCAMBIA1300-35S-Nos； And recombinant vector 1300-35S-MPK3-NOS be with

BnMPK3- 1-F (5 '-GGTACCTCTTCTCATTTCAGTCCCTCA-3 '),

BnMPK3-1-R (5’-GGATCCGGATATTTAGCCATTCATTCG-3’)

For primer target gene is amplified from cabbage type rape couples No. 9 cDNABnMPK3（This to primer be with The arabidopsis announced on NCBIAtMPK3（Gene ID: 823706）Coded sequence design）, and then tried according to gateway Agent box（Gateway LR Clonase II Enzyme Mix, Invitrogen Corporation）Operation, by purpose In gene cloning to the restriction enzyme site KpnI/BamHI of pCAMBIA1300-35S-Nos, 1300-35S-MPK3-NOS is obtained.

The present invention further provides describedBnMPK3Gene is improving rape to the application in the resistance of sclerotiniose, the present invention Reach the resistance for improving cabbage type rape to necrotrophic germ sclerotiniose by the overexpression gene.

Realize that the technology of the present invention is as follows：

Cabbage type rape cDNA library is created, with

BnMPK3-1- F (5 '-GGTACCTCTTCTCATTTCAGTCCCTCA-3 '),

BnMPK3-1- R (5 '-GGATCCGGATATTTAGCCATTCATTCG-3 ') is primer, double 9 from cabbage type rape Number cDNA library in PCR amplification go out cabbage type rapeBnMPK3CDNA sequence, be sequenced, it is found that this cDNA sequence is complete A length of 1299bp compares its Blast nucleotide in GenBank databases, finds this cDNA sequence and arabidopsis, tobacco etc. Transcription factor MPK3 have very high homology on nucleotide level.

In order to detect the reaction that MPK3 infects sclerotiniose, sclerotinite is used（Double No. 9 kinds separation from cabbage type rape, It is purchased from vegetable oil material institute of the Chinese Academy of Agricultural Sciences）It is inoculated with rape leaf, in inoculation 0,12,24,36,48 h then sampling is extracted respectively Blade total serum IgE, is then detected using real-time fluorescence quantitative PCRBnMPK3Expression quantity；With carrying 1300-35S-MPK3-NOS The Agrobacterium GV3101 of carrier（It is purchased from Shanghai and steps its bio tech ltd）Cabbage type rape is converted with flower-dipping method, is used Round pcr identifies transgenic line, is identified by Real-Time Fluorescent Quantitative PCR TechniqueBnMPK3Expression is significantly higher than non- The overexpression transgenic line of transgenic line.The disease-resistant evaluation experimental of sclerotiniose shows compared with non-transgenic strain,BnMPK3 It is overexpressed transgenic line and significantly improves resistance to sclerotinia sclerotiorum.

These the result shows thatBnMPK3Gene is with important application prospects in enhancing sclerotinia sclerotiorum resistance.

Advantages of the present invention：

1. the slave rape cDNA library employed in the present invention carrys out clone gene, reliability is high, speed is fast, efficient.

2. employed in the present invention it is agriculture bacillus mediated it is external dip osmosis conversion rape, speed is fast, it is at low cost, It is easy to operate.

3. clone obtains in the present inventionBnMPK3Gene provides new GENE SOURCES for other crop disease-resistant breedings；For Research how to improve other crops resistance to sclerotinia sclerotiorum have instruct reference function.

4. the MPK3 overexpression strains obtained after the 1300-35S-MPK3-NOS carriers conversion rape that the present invention is built, FromBnMPK3Function acquisition in terms of studied, beMPK3Disease-resistant functional study provide raw material, be of great significance.

5. the present invention is by rightMPK3A series of researchs of the overexpression strain in terms of anti-sclerotiniose, fully demonstrateMPK3Gene has positive effect in terms of Genes For Plant Tolerance sclerotiniose, to illustratingBnMPK3The biological function of gene is significant. Also it isMPK3In major oil crops（Rape）Application in terms of anti-sclerotiniose provides theory and practice foundation.Further to train It educates the rape variety with resistance to sclerotinia sclerotiorum and provides new breeding material.

6.BnMPK3The discovery of this sclerotinia sclerotiorum resistant gene, to excavate and identifying crop antibacterial core ospc gene, and The molecular mechanisms of action of further investigated antibacterial core ospc gene has important theory directive significance；In educating for the anti-sclerotiniose of crop Kind is put into practice, breed improvement and variety popularization all have important practical advice value.

Description of the drawings：

Fig. 1 sequencings obtainMPK3The 1-200 amino acid alignment result of sequence and other species MPK3.

Fig. 2 sequencings obtainMPK3The 201-376 amino acid alignment result of sequence and other species MPK3.

Each species of wherein Fig. 1 and Fig. 2 are abbreviated as：BnMAPK3 is the MPK3 sequences that sequencing obtains；Bj（Brassica juncea）：Leaf mustard；At （Arabidopsis thaliana）：Arabidopsis；Cb（Chorispora bungeana）：High mountain from Sub- mustard；Md（Malus domestica ）：Apple；Bp（Betula platyphylla）：White birch；Gh（Gossypium hirsutum）：Upland cotton；Sl（Solanum lycopersicum）：Tomato；Cm（Cucumis melo）：Muskmelon；Cs （Cucumis sativus）：Cucumber；Sp（Solanum peruvianum）：Lycopersicon peruvianum.Black region shows to be identical Amino acid, gray area show to be similar amino acid.

Fig. 3 isBnMPK3The reaction that sclerotinite is infected.

Fig. 4 is the building process schematic diagram of plant conversion carrier 1300-35S-MPK3-NOS.

Fig. 5 is rapeBnMPK3 It is overexpressed the identification of transgenic line；" just " it is 1300-35S-MPK3-NOS carriers PCR positive controls.#1 to #21 is respectively 21 overexpression transgenic lines." negative " is non-transgenic wild type control.

Fig. 6 is that qPCR is detected in transgenosis overexpression strain and non-transgenic WT strainBnMPK3The ratio of expression Compared with；WT is non-transgenic wild type control；O-2, O-5, O-6, O-8, O-18 and O-19 are independent overexpression transgenic line System.

Fig. 7 is non-transgenic WT strain compared with the resistance to sclerotinia sclerotiorum for being overexpressed transgenic line；WT turns base to be non- Because of wild type control；O-8 is transgenic line.

Fig. 8 is the comparison for being overexpressed transgenic line and non-transgenic WT strain sclerotiniose Spot expansion；WT is non- Transgenosis wild type control；O-2, O-6, O-8 are independentBnMPK3It is overexpressed transgenic line.

Specific implementation mode

Embodiment 1:BnMPK3The separation of gene is cloned

（1）BnMPK3The separation of gene and clone

With double No. 9 kinds in cabbage type rape（It is purchased from vegetable oil material institute of the Chinese Academy of Agricultural Sciences）As experiment material, item is grown Part is：Temperature is 20 ± 2 °C；Humidity is 60-90%；The daily photoperiod is 16 h of illumination 8 h dark；Intensity of illumination is 44 μ mol m^–2 s^–1。

The extraction of rape leaf total serum IgE and the synthesis of cDNA use 10 pillar Trizol kits of UNIQ-（It is purchased from Shanghai Handsome Bioisystech Co., Ltd）, use DNase I（Precious bioengineering (Dalian) Co., Ltd）It removes and is mixed in total serum IgE A small amount of DNA, finally with 1% agarose gel electrophoresis detect total serum IgE integrality.The synthesis of cDNA is inverted according to MMLV Record enzyme reagent kit（Pu Luomaige（Beijing）Bioisystech Co., Ltd）Operating instruction carry out, 50 μm of ol/L of primer Oligo(dT)18 （Thermo Fisher Scientific）.Then primer is used

BnMPK3-1- F (5 '-TCTTCTCATTTCAGTCCCTCA-3 ') and

BnMPK3-1- R (5 '-GGATATTTAGCCATTCATTCG-3 ') expands purpose band（This to primer be with The arabidopsis announced on NCBIAtMPK3（Gene ID: 823706）Coded sequence design）, then PCR product is cloned Into pMD18-T carriers（Precious bioengineering (Dalian) Co., Ltd）.Recycling, connection and the conversion reference UNIQ- of target fragment 10 pillar DNA plastic recovery kits（Sangon Biotech (Shanghai) Co., Ltd.）It is operated.Target fragment is carried with T The linked system of body is：4.6 μ L target fragments, 0.4 μ L pMD-18T carriers, 5 μ L Solution I（Precious bioengineering (Dalian) Co., Ltd）It is connected overnight at 16 DEG C.It is sent to Sangon Biotech's sequencing.

（2）BnMPK3Gene order structural analysis

Through sequencing, a length of 1299bp of the nucleotide sequence of this gene is found, containing there are one the open reading frame of overall length, make With ClustalX (Thompson et al., 1997）Program carries out the comparison discovery of a variety of amino acid sequences, this purpose item The MPK3 of band and arabidopsis, leaf mustard, high mountain ion mustard, apple, white birch, upland cotton, tomato, muskmelon, cucumber and Lycopersicon peruvianum exists There is very high homology on amino acid levels, it is consistent with the result compared on NCBI（Fig. 1 and Fig. 2）.

Embodiment 2:BnMPK3Reaction to sclerotinite

（1）The processing of sclerotinite

To Brassica Napus Seedling（When four one heart stages of leaf）Third leaf carries out sclerotiniose pathogen sclerotinite（From cabbage type rape Double No. 9 kinds（It is purchased from vegetable oil material institute of the Chinese Academy of Agricultural Sciences）Middle separation, activates through laboratory）Inoculation processing, processing before will plant Strain carries out 23 DEG C of constant temperature incubations 4~5 days, and then blade is placed in 24 h of dark surrounds moisturizing.Then use sclerotinite mycelia block into Row inoculation, control connect with sterile culture matrix.0 h, 12 h, 24 h, 36 h, 48 h samplings after inoculation respectively.

（2）The extraction of cabbage type rape RNA

Utilize plant Trizol（The handsome Bioisystech Co., Ltd in Shanghai）In reagent extraction steps 1 sclerotinite processing 0h, 12h, for 24 hours, the total serum IgE of 36h, 48h rear blade.

（3）Real-time quantitative PCR

Quantitative fluorescent PCR uses SYBR Green Realtime PCR Master Mix-Plus-kit（Treasured is raw Object engineering (Dalian) Co., Ltd）, using rape house-keeping geneBnUBC21WithBnTIP41As internal reference,BnUBC21 (ubiquitin-conjugating enzyme 21) is used as internal standard gene, and primer is by giving birth to work bioengineering (Shanghai) share Co., Ltd synthesizes.BnUBC21（Gene ID: 832645）Primer is：

F:5’- CCTCTGCAGCCTCCTCAAGT

R:5’- CATATCTCCCCTGTCTTGAAATGC

BnTIP41（Gene ID:AT4G34270）Due to lacking the sequence information of related gene, this hair in ncbi database The bright protein sequence using homologous gene in arabidopsis and the Brassica plants database announced（Brassica gene Database, BRAD）Carry out protein sequence comparison（BLASTP）To obtain homologous protein sequence of this reference gene in Chinese cabbage Row（Gene ID: Bra011516）And corresponding DNA sequence dna.Use the coding of the 5.0 software genes of Primer Premier Design specific primer in area（The areas DNA sequence dna poor information UTR provided due to database）, and apply Primer-BLAST (NCBI) in Brassica plants gene database（BRAD）Analysis is compared with primer specificity is carried out in rape est database, with Verify the specificity of primer.

BnTIP41Primer be:

5 '-TGAAGAGCAGATTGATTTGGCT-3 ' and

5 '-ACACTCCATTGTCAGCCAGTT-3 ',

BnMPK3The primer sequence of gene：

BnMPK3-2-F：5 '-ACCAGGGCTTGTCTGAGGA-3 ' and

BnMPK3-2-R： 5´- AATCGCAGTTGGCGTTCA- 3´.

（This is to be drawn with the quantitative fluorescent PCR specificity for the nucleotide sequence design being sequenced in embodiment 1 to primer Object）Fluorescent quantitation reaction system is 20 μ L：Contain 10 μ L of SYBR Mix, forward and reverse primer（10 µmol/L）Each 0.8 μ L, The 2 processed sterile water of μ L and DEPC of template.Amplification condition is：95 DEG C, 30sec；Then 95 DEG C of 5s, 60 DEG C of 30 s, 72 DEG C 27s, 40 cycles；It is each to be circulated in 72 DEG C of renaturation ends progress fluoroscopic examinations.It is first to heat to 95 DEG C after reaction, then drops To 72 DEG C, then 95 DEG C are to slowly warm up to, record the variation of fluorescence signal, obtains the melting curve of amplified production.Every group of experiment is equal It completes three biology to repeat, each biology repeats at least to do technology repetition three times.DetectionBnMPK3Respectively in rape nuclear disk The Expression variation of the blade 0-48 h of bacterium induction.

7300 System- of software carried using ABI (Applied biosystems) model QPCR7300 instrument SDSS hell optimize internal standard gene and target gene PCR reaction conditions, it is special to be allowed to amplified production.This experiment is with rapeBnUBC21WithBnTIP41For internal standard gene, the Ct values of internal standard gene and target gene are measured respectively, take its average value, with comparing Ct methods obtain DCt of the target gene to internal standard gene, then using the blade of water process as reference（Its value is converted into 1）Respectively The DDCt for obtaining other processing, the Relative Expression values of target gene are estimated finally by 2-DDCt, and find out systematic error.

The results show that when sclerotinite infects 12h,BnMPK3Transcriptional level increase 5.06 times, when sclerotinite infects 48h When,BnMPK3Expression quantity compared to be not inoculated with nuclear disk bacteria strain improve 8.98 times（Fig. 3）, illustrate rapeBnMPK3To sclerotinite Infect and have significant response.

Embodiment 3:It is overexpressedBnMPK3The acquisition of transgene rape plant

（1）Plant over-express vector is built

According to the CaMV 35S sequences announced on NCBI（Gene ID:AJ007626）Use Primer Premier 5.0 Software Design primers

5 '-GAATTCTTAATTAAGAGCTCGCATGCC-3 ' and

5 '-GGTACCGTCCCCGTGTTCTCTCCAA-3 ', using the means of PCR from pEGAD carriers（It is purchased from precious biological work Journey (Dalian) Co., Ltd）Middle amplification obtains CaMV 35S segments, is subsequently attached to pCAMBIA1300 carriers（It is purchased from north Prosperity biotechnology Co., Ltd of Jing Ding states）EcoRI/KpnI restriction enzyme sites；Simultaneously according to the CaMV announced on NCBI Nos sequences（Gene ID: AJ007624）Use 5.0 software Design primers of Primer Premier

5 '-GGATCCGAATTTCCCCGATCGTTCAA ' -3 ' and

5 '-AAGCTTGATCTAGTAACATAGATGACACCGC-3 ' are expanded from pEGAD carriers with PCR and are obtained CaMV Nos segments are connected to pCAMBIA1300 carriersBamHI/HinDIII restriction enzyme sites, then obtain pCAMBIA1300- 35S-Nos carriers.Restriction endonuclease EcoRI, KpnI used,BamHI andHinDIII is purchased from precious bioengineering (Dalian) limited public affairs Department, digestion condition are 37 DEG C of standing 12h.

Then design primer（With the arabidopsis announced on NCBIAtMPK3Coded sequence（Gene ID: 823706）If Meter）

BnMPK3-1-F ：5 '-GGTACCTCTTCTCATTTCAGTCCCTCA-3 ' and

BnMPK3-1-R：5 '-GGATCCGGATATTTAGCCATTCATTCG-3 ', using cabbage type rape cDNA as template into Row PCR amplification obtains the purpose segment of a length of 1299kb, is connected to the KpnI/BamHI of pCAMBIA1300-35S-Nos On restriction enzyme site, reaction system is：I. 1 μ L, 10*Hbuffer1 μ L, DNA 1ul (2 μ g) of KpnI, 7 μ L of ddH20；II. BamHI 1 μ L, 10*Kbuffer1 μ L, DNA 1ul (2 μ g), 7 μ L of ddH20 place it in 37 DEG C of standing 12h.To create To cabbage type rapeBnMPK3Transgenic over expression carrier 1300-35S-MPK3-NOS, it is mould containing moisture resistance in its area T-DNA The selection markers of element,BnMPK3The promoter of overexpression is 35s promoters（Fig. 4）.

（2）The genetic transformation of rape

A. carrier 1300-35S-MPK3-NOS is transferred to Agrobacterium GV3101（It is purchased from Shanghai and steps the limited public affairs of its biotechnology Department）, disseminate rape flower three times, five minutes every time.Disseminating step is：

B. it cultivates and contains in the test tube of the LB liquid medium containing rifampin, gentamicin, kanamycins (50mg/mL) There is the Agrobacterium GV3101 of recombinant plasmid, then contains above-mentioned three kinds of antibiotic in 10% ratio access（50mg/mL）LB liquid The triangular flask of body culture medium makes the final concentration of OD600 of Agrobacterium culture solution be about 2.0 or so.

C. conversion buffer solution is added in culture medium：

0.1% Silwet-L77

2ng/L 6-BA

8mg/L acetosyringones

It is obtained after the entire bud part that do not open of rape is immersed in 1300-35S-MPK3-NOS carriers conversion GV3101 Agrobacterium solution in 3-5min, while gently shaking makes bud come into full contact with agrobacterium liquid.The bud impregnated is inserted in sheep Mulberry paper bag for 24 hours, to keep bud to have higher humidity, avoids bud from being exposed to the sun in excessive sunlight, prevents Agrobacterium from losing Activity.

D. step c is repeated every two days, and entire bud is sleeved on 48h in sheepskin paper bag.

E. after dip-flower, remove the sheepskin paper bag on inflorescence, shear the side shoot inflorescence newly born and by dip-flower The bud that its top of inflorescence is newly sprouted, and anchor line (string) label, difference weight are carried out to the inflorescence for disseminating Agrobacterium using colored silk thread The structure of group plasmid is marked into row label.

F. normal to pour culture plant, and newborn side shoot inflorescence and newborn bud are wiped out in timing, until seed at Stop irrigating when ripe, harvest dry seeds, the screening of transformant is carried out by the selected marker carried on recombinant plasmid, passes through this side Method filters out largelyBnMPK3Overexpression transformant.

（3）Transfer-gen plant is identified

The hygromycin of 25mg/L is first used after the seed sprouting of harvest（It is purchased from Beijing Baeyer enlightening Bioisystech Co., Ltd） Screening, chooses the plant of hygromycin, carries out label distinction respectively（Number is from #1 to #21）, extract genome, with PCR into Row identification, primer used are

BnMPK3-1-F ：5 '-TCTTCTCATTTCAGTCCCTCA-3 ' and

BnMPK3-1-R ：5 '-GGATATTTAGCCATTCATTCG-3 ',

Control is non-transgenic WT lines（WT）, it is negative control group；Positive control is one containing the gene Plasmid.

Testing result shows（Fig. 5）, positive control and #1-#3, #5-#8, #10-#15, #17-#19 this 16 transformed plants The electrophoretic band of expected size can be amplified（952bp）, and negative control shows transgene rape then without electrophoretic band Foreign gene DNA segments are contained in genome.

（4）It is overexpressedBnMPK3The qPCR of transgene rape is identified

For determinationBnMPK3In rape whether overexpression, using qPCR methods to step（3）Identify that is obtained turns Gene plant is analyzed,BnMPK3Amplimer be

BnMPK3-2-F ：5'-ACCAGGGCTTGTCTGAGGA-3' and

BnMPK3-2-R：5'-AATCGCAGTTGGCGTTCA-3'（This is to the core that primer is to be sequenced in embodiment 1 The quantitative fluorescent PCR specific primer of nucleotide sequence design）, reference gene isBnTIP41, amplimer 5 '- Work synthesis is given birth in TGAAGAGCAGATTGATTTGGCT -3 ' and 5 '-ACACTCCATTGTCAGCCAGTT-3 ', Shanghai.PCR journeys Sequence：Then 95 DEG C of pre-degeneration 30S are recycled through 40（95℃ 10s、57℃ 10s、72℃ 26s）.

By step（3）Identify the plant number front plus O that obtained transfer-gen plant analysis is overexpressed（The abbreviation of over）.

As shown in fig. 6, O-2, O-5, O-6, O-8, O-18 and O-19 strainBnMPK3Expression is all remarkably higher than non- The wild type control of transgenosis, for expression quantity at 3 times or more, the expression quantity of O-18 strains reaches 7 times of WT strain.Show These strains are independentBnMPK3It is overexpressed transgenic line.

Embodiment 4：BnMPK3It is overexpressed the evaluation of the anti-sclerotiniose of transgene rape

One heart stage of four leaves of PCR test positive turns in Example 3BnMPK34th true leaf of positive plant into Row Vitro Inoculation Technique.Blade, which is cut, to be placed in ceramic whiteware disk, and each rotaring gene plant blade is placed side by side one with non-transgenic reference In disk, one layer of blade lower berth wet gauze takes freshly prepared φ 5mm mycelia blocks, has the face-down of mycelia, be inoculated in middle part of blade It is moved slightly away from the position of master pulse, two mycelia blocks are inoculated with per leaf.Pan Kou is coverd with ventilated membrane and is conducive to moisturizing, then sets 22 DEG C of dark Culture.

12 h start to observe and record mycelia that a situation arises after cultural hypha, and 24 h start the life of observational record bacterial plaque after culture Long amount, it is primary every 24 h, until mycelia grows to tooth in vitro edge.

Isolated leaf inoculation qualification result shows that resistance to sclerotinia sclerotiorum is remarkably reinforced in transfer-gen plant.As shown in Figure 7 and Figure 8, From persistently being carried out to blade within 0h to 72 hours from inoculation, it is found that sclerotinite scab existsBnMPK3It is overexpressed in transgenic line Growth area is more much smaller than in non-transgenic strain, and the area of injury tissue also wants small.It can according to the growth area of sclerotinite To find, WT lines are soft addle it is dead be happened at after sclerotinite is inoculated with 18 h, and grow during 30-54 h it is rapid, 54 Growth area reaches maximum when h.BnMPK3The time of infection for being overexpressed transfer-gen plant is more late than wild-type strain, bacterial plaque diffusion Rate and wild-type strain are not much different, and because of its time of infection evening, after sclerotinite is infected 72 hours reaching maximum infects area. It follows thatBnMPK3Resistance of the rape leaf to sclerotiniose can effectively be enhanced, and may be to resist to invade type to the resistance of sclerotiniose.

SEQUENCE LISTING

<110>Jiangsu University

<120>The application of cabbage type rape gene BnMPK3 and its anti-sclerotinia sclerotiorum

<130>The application of cabbage type rape gene BnMPK3 and its anti-sclerotinia sclerotiorum

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1299

<212> DNA

<213>Cabbage type rape（Brassica napus）

<400> 1

tcttctcatt tcagtccctc atctaactgt aatataatta tatatataaa ggagtcacac 60

aataccttca gattcactat acctcaaagc acaacaagtc aagcttgaag ttgacttagc 120

gcgtcatcga ttgaaagaga gagagagaga gagatgaaca acgccggcgg ccaatatcca 180

gattttccgg cggtgcagac gcacggagga cagttcataa gctacgatat cttcggtagt 240

ttattcgagg tcacatccaa gtaccgtcct ccgattgttc caatcggtcg gggcgcatac 300

ggcatcgttt gctctgtgtt ggattcggag acgaacgagc ttgtggcgat gaagaagata 360

gccaacgcct ttgataatca catggacgcc aagcgtacac ttcgcgagat caagcttctt 420

cgtcatctcg atcatgaaaa catcatagct ataagagatg tggttcctcc accacttaga 480

agagagttca gtgatgttta tatcgccact gggttaatgg acactgatct tcatcaaatc 540

atcagatcca accagggctt gtctgaggaa cactgtcagt acttcctgta ccagcttctt 600

cgagggctca agtacatcca ctcggctaaa gttatccaca gggatctaaa gcctagcaat 660

cttctcctga acgccaactg cgatttaaag atctgtgatt ttggtcttgc tagacccact 720

tcagagaacg agtttatgac tgagtatgtt gttaccagat ggtatagagc acctgagctt 780

ctgctcaact catctgatta cacagctgct atagatgttt ggtctgttgg ttgtatcttt 840

atggagctca tgaacagaaa gcctttgttc cctggtaaag accatgtcca tcagatgcgc 900

ttattgacag agttgcttgg cacgccgaca gaatcagatc tcgggtttac acacaacgag 960

gatgccaaaa gatacatccg gcagcttccc aacttccctc gccaaccgtt agctaaactg 1020

ttctctcatg ttaactcatt ggccattgat ctagttgaca gaatgttgac gtttgacccc 1080

aacaaaagaa tcactgttga agaagctctg aatcacccgt acctagccaa gttgcacgac 1140

ccgaatgatg agccaatctg tctaaagcca ttctctttcg actttgaaca acaacctcta 1200

gacgaggaac agataaaaga gatgatctac agggaagcca ttgcactcaa tccaacatat 1260

gcttagaaga gcagcaaccg aatgaatggc taaatatcc 1299

<210> 2

<211> 370

<212> PRT

<213>Cabbage type rape（Brassica napus）

<400> 2

Met Asn Asn Ala Gly Gly Gln Tyr Pro Asp Phe Pro Ala Val Gln Thr

1 5 10 15

His Gly Gly Gln Phe Ile Ser Tyr Asp Ile Phe Gly Ser Leu Phe Glu

20 25 30

Val Thr Ser Lys Tyr Arg Pro Pro Ile Val Pro Ile Gly Arg Gly Ala

35 40 45

Tyr Gly Ile Val Cys Ser Val Leu Asp Ser Glu Thr Asn Glu Leu Val

50 55 60

Ala Met Lys Lys Ile Ala Asn Ala Phe Asp Asn His Met Asp Ala Lys

65 70 75 80

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

85 90 95

Ile Ile Ala Ile Arg Asp Val Val Pro Pro Pro Leu Arg Arg Glu Phe

100 105 110

Ser Asp Val Tyr Ile Ala Thr Gly Leu Met Asp Thr Asp Leu His Gln

115 120 125

Ile Ile Arg Ser Asn Gln Gly Leu Ser Glu Glu His Cys Gln Tyr Phe

130 135 140

Leu Tyr Gln Leu Leu Arg Gly Leu Lys Tyr Ile His Ser Ala Lys Val

145 150 155 160

Ile His Arg Asp Leu Lys Pro Ser Asn Leu Leu Leu Asn Ala Asn Cys

165 170 175

Asp Leu Lys Ile Cys Asp Phe Gly Leu Ala Arg Pro Thr Ser Glu Asn

180 185 190

Glu Phe Met Thr Glu Tyr Val Val Thr Arg Trp Tyr Arg Ala Pro Glu

195 200 205

Leu Leu Leu Asn Ser Ser Asp Tyr Thr Ala Ala Ile Asp Val Trp Ser

210 215 220

Val Gly Cys Ile Phe Met Glu Leu Met Asn Arg Lys Pro Leu Phe Pro

225 230 235 240

Gly Lys Asp His Val His Gln Met Arg Leu Leu Thr Glu Leu Leu Gly

245 250 255

Thr Pro Thr Glu Ser Asp Leu Gly Phe Thr His Asn Glu Asp Ala Lys

260 265 270

Arg Tyr Ile Arg Gln Leu Pro Asn Phe Pro Arg Gln Pro Leu Ala Lys

275 280 285

Leu Phe Ser His Val Asn Ser Leu Ala Ile Asp Leu Val Asp Arg Met

290 295 300

Leu Thr Phe Asp Pro Asn Lys Arg Ile Thr Val Glu Glu Ala Leu Asn

305 310 315 320

His Pro Tyr Leu Ala Lys Leu His Asp Pro Asn Asp Glu Pro Ile Cys

325 330 335

Leu Lys Pro Phe Ser Phe Asp Phe Glu Gln Gln Pro Leu Asp Glu Glu

340 345 350

Gln Ile Lys Glu Met Ile Tyr Arg Glu Ala Ile Ala Leu Asn Pro Thr

355 360 365

Tyr Ala

370

Claims (4)

1. a kind of recombinant vector 1300-35S-MPK3-NOS, which is characterized in that the carrier includes shown in SEQ ID NO.1BnMPK3The nucleotide sequence of gene.

2. applications of the recombinant vector 1300-35S-MPK3-NOS as described in claim 1 in anti-sclerotinia sclerotiorum.

3. application according to claim 2, which is characterized in that include the following steps：Build recombinant vector 1300-35S- MPK3-NOS；Obtained recombinant vector is transferred to Agrobacterium GV3101, cabbage type rape is converted with flower-dipping method, using round pcr Identify transgenic line；It is identified by Real-Time Fluorescent Quantitative PCR TechniqueBnMPK3Gene expression dose；Recombinant vector 1300- 35S-MPK3-NOS transfectionsBnMPK3The rape leaf for being overexpressed transgenic line shows the resistance to sclerotiniose.

4. a kind of application of the rape BnMPK3 genes as shown in SEQ ID NO.1 in anti-sclerotinia sclerotiorum.