CN109777810A - PUB41 gene is improving the application in graw mold of tomato and Resistance to bacterial wilt as negative regulatory factor - Google Patents

Abstract

Application the invention discloses PUB41 gene as negative regulatory factor in raising graw mold of tomato and Resistance to bacterial wilt, the nucleotide sequence of the protein-coding region of the PUB41 gene is as shown in SEQ ID NO.1.The present invention obtains tomato PUB41 gene editing mutant using CRISPR/Cas9 gene editing technology, it was found that the mutant can significantly increase tomato to the resistance of gray mold and bacterial wilt, PUB41 gene is demonstrated as negative regulatory factor and is improving the purposes in graw mold of tomato and Resistance to bacterial wilt, can be used for the breeding of botrytis resistant and bacterial wilt tomato variety.

Description

PUB41 gene is improving graw mold of tomato and Resistance to bacterial wilt as negative regulatory factor In application

Technical field

The present invention relates to field of biotechnology more particularly to PUB41 gene as negative regulatory factor in raising tomato gray mould Application in disease and Resistance to bacterial wilt.

Background technique

With the progress and development of China's modern agriculture, vegetables industry also flourishes.However, since plant is exposed to carefully In the pathogenic microorganisms environment such as bacterium, virus and fungi, pest and disease damage causes serious harm to the yield and quality of vegetables.Together When, Global climate change has been further exacerbated by the generation of biotic and abiotic stress, and facilities vegetable disease occurs further Frequently.

Tomato (Solanum lycopersicum L.) is worldwide industrial crops, is that current facility cultivation area is maximum One of vegetable variety, but various bacillary, viral and fungal diseases occur very rampant, cause to tomato production Extremely serious economic loss.Graw mold of tomato and bacterial wilt are extremely incident two kinds of typical diseases in facility cultivation.

Graw mold of tomato is by the microbial fungal disease of Botrytis cinerea, is a kind of worldwide disease, while being also tomato On a kind of Major Diseases.Graw mold of tomato can all occur in various protecting fields, and spread speed is fast, especially in winter-spring season Section low temperature, the interior morbidity of greenhouse of high humidity are more serious, typically result in underproduction 20%-40%.

Bacterial wilt of tomato is a kind of bacteria type disease, and there is serious hair in each province (city) tomato planting region in south China It is raw.The disease is that the worldwide crushing soil of one kind as caused by Ralstonia solanacearum passes bacteriosis, in high temperature, high humidity Under the conditions of be easy to break out, be distributed widely in the torrid zone, subtropical zone and Temperate Region in China, or even in part low temp area it is again seen that green withered The presence of bacterium brings great threat to global production estimation.The disease is difficult to control once occurring, and often results in work Object large area wilts dead or even has no harvest, and leads to tomato yield degradation, seriously restricts the development and economy of tomato industry The raising of benefit.

Currently, the upper a large amount of prevention and treatments for carrying out gray mold and bacterial wilt using chemicals of production, lead to have drug resistance Bacterial strain is filtered into as dominant group, progress mass propagation, to make under the drug effect of chemical agent under the action of natural selection Drop, or even lose.Also, chemicals prevention and treatment causes serious harm to environment, destroys ecological environment, and it is strong to endanger the mankind Health.Some " the bad genes " of crops itself can be knocked out by gene editing technology, achieving the purpose that rogue, it is excellent to deposit.And And gene editing technology is not transferred to foreign gene, only modifies gene existing for crop inside.In the plant finally obtained In strain, some foreign constituents are not remained, have the advantages that be no different with routine mutagenesis kind, therefore in the production of crop improvement Using upper safer.It, can be with by gene editing technology currently, CRISPR/Cas9 gene editing technology comparative maturity The negative regulator gene of gray mold and bacterial wilt in plant is knocked out, so that control accurate is carried out to graw mold of tomato and bacterial wilt, Realize germplasm innovation.Therefore, it finds and studies the key gene that there is negative regulation function to graw mold of tomato and Resistance to bacterial wilt It is of great significance for plant disease-resistant breed improvement.

PUBs family is a kind of E3 ubiquitin ligase containing U-box structural domain, other than conserved domain U-box, also There are ARM repetitive sequence, the other structures such as kinases domains.PUB gene family is in regulating growth of plants, biology and the abiotic side of body It is of great significance in compeling.Arabidopsis AtPUB4 gene can influence the growth and development of pollen tapetal cell, and pub4 mutant goes out Existing tapetum is not exclusively degenerated and the abnormal phenotype of pollen wall outer membrane (Wang H etc., " The Arabidopsis U-box/ ARM repeat E3ligase AtPUB4influences growth and degeneration of tapetal cells,and its mutation leads to conditional male sterility.”The Plant Journal,2013,74(3):511-523).In addition to this, U-box ubiquitin ligase by adjust ABA signal path and ABA synthesis, to sprout in seed and play an important role in Leaf Senescence.The mutant of arabidopsis pub9 is sprouted in seed , abi3pub9 double-mutant sensibility forfeiture to ABA sensitive to ABA is shown during hair.Illustrate U-box ubiquitin ligase PUB9 functions (Raab S etc., " Identification of a novel E3ubiquitin in the upstream of ABI3 ligase that is required for suppression of premature senescence in Arabidopsis.Plant Journal for Cell&Molecular Biology,2010,59(1):39-51).In capsicum On, CaPUB1 is as E3 ubiquitin ligase negative regulation plant dehydration and high-salt stress.CaPUB1 promotes 26S by ubiquitination pathway The degradation of proteasome subunit RPN6, in the transgenic line for being overexpressed CaPUB1, the significant gene RD29a of drought stress Expression quantity obviously raise (ChoS.K etc., " Heterologous Expression and Molecular and Cellular Characterization of CaPUB1Encoding a Hot Pepper U-Box E3Ubiquitin Ligase Homolog.Plant Physiology,2006,142(4):1664-1682).Also, the E3 ubiquitin containing U-box structural domain Ligase reacts initial from plant immune, i.e., pathogen recognize downstream signaling pathway it is each during, play just Regulation or negative regulation effect.Under the induction of flagellin, arabidopsis PUB22/23/24 expression quantity rises.In pub22/23/24 In Trimutant, by PTI immunologic pathways caused by enhancing PAMP, to improve the resistance to bacterial leaf spot (" the Negative Regulation of PAMP-Triggered Immunity by an such as Trujillo M E3Ubiquitin Ligase Triplet in Arabidopsis.Current Biology,2008,18(18):1396- 1401)。

The function of tomato PUB gene family also rarely has people's report, and function of the PUB41 in tomato immune defense is not ground also Study carefully report.Therefore, resistance mechanism of the research tomato PUB41 gene in gray mold and bacterial wilt has theory and actual application valence Value.

Summary of the invention

It is new in raising graw mold of tomato and Resistance to bacterial wilt as negative regulatory factor that the present invention provides PUB41 genes Purposes provides foundation to cultivate the tomato variety of botrytis resistant and bacterial wilt.

Specific technical solution is as follows:

Application the present invention provides PUB41 gene as negative regulatory factor in raising graw mold of tomato resistance, it is described For the nucleotide sequence of the protein-coding region of PUB41 gene as shown in SEQ ID NO.1, encoding histone section length is 1689bp, Complete genome sequence is the same as shown in SEQ ID NO.1.

The albumen of PUB41 gene coding is the E3 ubiquitin ligase containing U-box structural domain, by 560 amino acid groups At, sequence as shown in SEQ ID NO.2, can be degraded substrate protein by ubiquitination pathway with specific recognition target proteins.

The present invention utilizes CRISPR/Cas9 gene editing technology, carries out sequence analysis to tomato PUB41 gene, searches PAM The sequence definition of 20 bp before NGG is sgRNA by sequence, is selected and is located on gene protein code area and has high special The sgRNA sequence of property.The DNA sequence dna such as SEQ ID of the sgRNA of the selectively targeted tomato PUB41 gene protein code area Shown in NO.3.

The present invention is tested by external ubiquitination and is found, PUB41 can pass through ubiquitin with specific recognition tomato protein kinase Change approach degradation substrate protein；Tomato protein kinase is the key gene for defending ash arrhizus bacteria, is planted after silencing tomato protein kinase Strain declines the defence capability of graw mold of tomato.PUB41 defends the key gene of ash arrhizus bacteria by ubiquitination tomato, to cut Resistance of the weak tomato to gray mold.

So further, the albumen of PUB41 gene coding is as negative regulatory factor in improving graw mold of tomato resistance Application, the amino acid sequence of the albumen is as shown in SEQ ID NO.2.

Further, the albumen with tomato protein kinase by specifically binding, and then negative regulation tomato is to gray mold Resistance.

The negative regulation refers to that PUB41 gene by specifically binding with tomato protein kinase, passes through ubiquitination pathway Degradation immune defense key protein tomato protein kinase, to weaken tomato to the resistance of gray mold.

PUB41 gene is improving the application in bacterial wilt of tomato resistance, the egg of the PUB41 gene as negative regulatory factor The nucleotide sequence of white code area is as shown in SEQ ID NO.1.

The present invention also provides a kind of methods of cultivation botrytis resistant and the tomato of bacterial wilt, comprising:

(1) target fragments containing PAM structure are chosen in the protein-coding region of tomato PUB41 gene, with target fragments PAM 20 bases are foundation before structure, carry out design of primers, construct CRIPR/Cas9 carrier；

(2) the Agrobacterium genetic engineering bacterium of the CRIPR/Cas9 carrier of (1) containing step is constructed；

(3) step (2) genetic engineering bacterium is converted into tomato cotyledon, obtain without external source Cas9 albumen and stablizes heredity Mutants homozygous strain.

Further, before the target fragments PAM structure nucleotide sequence of 20 bases as shown in SEQ ID NO.3. The PAM structure is NGG, and N represents any base.

Further, the nucleotide sequence such as SEQ ID NO.4 and SEQ of the primer of the CRISPR/Cas9 carrier is constructed Shown in ID NO.5.

Compared with prior art, the invention has the following advantages:

The present invention obtains tomato PUB41 gene editing mutant using CRISPR/Cas9 gene editing technology, it is found that this is prominent Variant can significantly increase tomato to the resistance of gray mold and bacterial wilt, it was demonstrated that PUB41 gene is being mentioned as negative regulatory factor Purposes in high graw mold of tomato and Resistance to bacterial wilt can be used for the breeding of botrytis resistant and bacterial wilt tomato variety.

Detailed description of the invention

Fig. 1 is the sequencing analysis figure of the catastrophe point of the pub41#1 homozygous deletion mutant obtained in embodiment 2；

Wherein, "-" indicates base deletion；Compared with the Lycopersicon esculentum without gene editing, mutant is in the position of sgRNA Set generation base deletion；It will be known as compareing without the Lycopersicon esculentum of gene editing below.Pub41#1 is than compareing few two alkali Base.

Fig. 2 is control and the sick grade index column after the inoculation of pub41#1 homozygous deletion mutant gray mold 2 days in embodiment 3 Shape figure；

Wherein, it falls ill more serious, sick grade index is higher；Open tubular column represents plain edition tomato, and it is pure that twill column represents pub41#1 Close deletion mutant plant；Lowercase a, b represent the significant difference between different disposal in 5% level；The result shows that control Sick grade index be significantly higher than mutant material.

Fig. 3 is control and the incidence of leaf platform after the inoculation of pub41#1 homozygous deletion mutant gray mold 2 days in embodiment 3 Expect blue colored graph；

Wherein, spot represents dead cell quantity, and the spot of Lycopersicon esculentum is more than mutant material, illustrates control morbidity than prominent Variant material is serious.

Fig. 4 is Lycopersicon esculentum and the phenotypic map after the inoculation of pub41#1 homozygous deletion mutant bacterial wilt 7 days in embodiment 4； The result shows that control morbidity is more serious than mutant material.

Fig. 5 is that Lycopersicon esculentum refers to the sick grade after the inoculation of pub41#1 homozygous deletion mutant bacterial wilt 7 days in embodiment 4 Number cylindricality；

Wherein, it falls ill more serious, sick grade index is higher；Open tubular column represents plain edition tomato, and it is pure that twill column represents pub41#1 Close deletion mutant plant；Lowercase a, b represent the significant difference between different disposal in 5% level.The result shows that control Sick grade index be significantly higher than mutant material.

Fig. 6 is the result figure of external ubiquitination test in embodiment 5；

External ubiquitination system includes E1, E2, E3, substrate protein and ubiquitin molecule (ubiqutin)；Wherein, E1 is indicated Ubiquitin kinase；E2 indicates ubiquitin binding enzyme；PUB41 has GST label as E3 ubiquitin ligase；Tomato protein kinase is made For E3 substrate specificity albumen, MBP label is had, ubiquitin molecule has HA label；Different label proteins can be detected by different antibodies； Albumen is changed by molecular weight after ubiquitination, a ubiquitin 8.5kDa, and albumen may be by one, two or more ubiquitin marks Note, so will appear ubiquitination band as shown in the figure；Without E3 ubiquitin ligase as negative control in system.

Specific embodiment

The invention will be further described combined with specific embodiments below, and what is be exemplified below is only specific implementation of the invention Example, but protection scope of the present invention is not limited only to this.Unless otherwise specified, technological means used in embodiment is this field Known to technology people, raw materials used, kit is commercial goods.

Following tomato varieties used in the examples are tomato conventional variety CR (Condine Red), below will be without The Lycopersicon esculentum of gene editing is known as compareing.

The building of 1 mutant material C RISPR/Cas9 carrier of embodiment

(1) in https: inputting tomato PUB41 gene order on the //website solgenomics.net/tools/blast/ Number Solyc06g051090 finds its DNA sequence dna as shown in SEQ ID NO.6, inputs http: // The website crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR, it is high to find out on score score, and G/C content > 40%, CRISPR primer, following institute are designed in one section of 20bp base sequence (SEQ ID NO.3) containing NGG in the area CDS in position Show:

Primer (SEQ ID NO.4): 5 '-GATTGCACTTGACAT TTTACCTCTG-3 ' before CRISPR-PUB41-

Primer (SEQ ID NO.5): 5 '-AAACCAGAGGTAAA ATGTCAAGTGC-3 ' after CRISPR-PUB41-

(2) above-mentioned preceding primer and each 5 μ l of rear primer are taken, after mixing, is annealed into double-strand with PCR instrument, is oriented to RNA as single (sgRNA)。

(3) by intermediate vector pMD18-T through BbsI single endonuclease digestion, it is connected after purification with the sgRNA for being annealed into double-strand, is connected with T4 After connecing enzyme connection, 42 DEG C of thermal shocks convert coated plate, and pMD18-T carrier resistance is ammonia benzyl mycin.

(4) monoclonal colonies are chosen, with primer before CRISPR (SEQ ID NO.4): GATTGCA Primer (SEQ ID NO.7) after CGGCGGCGGTATTTGTGG and carrier: CTACTTATC GTCATCGTCTTTG carries out PCR and tests Card.

(5) after sequencing is correct, upgrading grain is connected to whole carrier pCAMBIA1301 after Hind III and Kpn I double digestion On.PCAMBIA1301 carrier resistance is kanamycins.Correct plasmid is sequenced to shock by electricity into GV3101 Agrobacterium competence, obtains The carrier of its CRISPR material must be can be used for constructing.

The preparation and identification of embodiment 2PUB41 gene mutation body material

(1) aseptic seedling is cultivated

Tomato seeds impregnate (or being shaken 6-8 hours with 28 DEG C of 200rpm/min of shaking table) with tap water, then with 75% alcohol Disinfection 30 seconds sterilizes in 10%NaClO 15 minutes (with 28 DEG C of 200rpm/min of shaking table) later, and sterile purified water rinses 3 times And sterilizing vessel are transferred to, it is inoculated in 1/2Murashige and Skoog (MS) culture medium.Cultivated under 25 DEG C of dark conditions to Germination.

(2) prepare explant

6 days after germination, the cotyledon of aseptic seedling is cut with knife, cotyledon has a bit of petiole, is placed in accompanied culture Preculture 1 day (being protected from light, overnight, accompanied culture overlong time, which is easy to cause, to be infected excessively) in base.

(3) Agrobacterium is infected

It will be cultivated in embodiment 1 to the Agrobacterium bacterium solution centrifugation of mid-log phase (OD600 ≈ 1.0), remove supernatant, be added outstanding Floating culture medium MS0.2, shakes up.Cotyledon explant is transferred in the sterilizes culture dish of MS0.2, pours into the bacterium solution to have suspended extremely It is dark inoculation 4-5 minutes lower, and shake gently culture dish.Explant is transferred in sterilizing filter paper, remaining bacterium solution is blotted, Cotyledon explant reverse side is transferred to upward on accompanied culture base, 22 DEG C are protected from light co-cultivation 2 days.

(4) selection culture, regeneration

After co-cultivation, by explant being transferred on MRS1 (2Z+) carefully, culture in MRS2 (0.2Z+) is transferred to after 2-3 weeks, The material of pollution is cleared up in time during selection culture, until growing regeneration bud.

(5) regeneration bud is taken root, is transplanted

At long to 1 centimetre or so of bud to be regenerated, bud is cut (it can not cut, in order to avoid injury position of taking root), it is put into and takes root It takes root in culture medium.Hardening is carried out to good, the long transformation seedlings to 5cm or so of taking root after 2 weeks, is transplanted after surviving into flowerpot, Obtain T₀For gene editing material.

(6) tomato pub41#1 Mutants homozygous material identification

T is extracted using CTAB method₀Genomic DNA for plant and using it as template, before the DNA sequence dna comprising sgRNA The following primer of design about at 200bp afterwards, carries out PCR amplification sequence verification:

Primer (SEQ ID No.8) before verifying: GCTCTTTGTGGAACCTCATG

Primer (SEQ ID No.9): CCTATCACAGTTAGTAGCCA after verifying

After the sequence amplification comprising 20bp sgRNA of above-mentioned selection is come out, sequencing company is sent to be sequenced, sequencing result benefit It is compared with DNAMAN software.It chooses sgRNA sequence base deletion occurs and is sequenced to show unimodal plant, carries out selfing breeding, Obtain the seed in T0 generation.

The seed in T0 generation is after planting obtained into T1 for plant, using above method detection T1 for the sgRNA sequence alkali of plant Base editor's situation.Meanwhile using primer (SEQ ID NO.7) after primer before CRISPR (SEQ ID NO.4) and carrier to DNA into Row PCR amplification detects T1 for plant and whether contains Cas9.It chooses and is free of Cas9, but the T1 that morphs of sgRNA is for plant, Greenhouse breeding, selfing, which obtains, is free of foreign gene Cas9, and stablizes the T1 of heredity for 1 strain of Mutants homozygous plant, name For pub41#1, gene editing site is as shown in Figure 1.

Pub41#1 is than compareing few two bases.Breeding is further selfed to above-mentioned strain, obtains T1 for seed.By T1 generation Seed after planting, obtains and is free of foreign gene Cas9, and the T2 for stablizing heredity that sgRNA morphs is for plant.

Following embodiment is tested for homozygous lines as material using T2.

3 tomato pub41#1 gene editing mutant of embodiment influences gray mold resistance

Grey mold body inoculation experiments: botrytis cinerea (BO5-10) culture V8 solid medium (36%V8 fruit juice, 0.2% CaCO3,2% agar powder) it is cultivated 15 days or so under the conditions of being protected from light for 16 DEG C, after covering with spore in ware, 4 DEG C are kept in dark place.Make Mycelia block is put into inoculation medium (1% peptone, 4% 1 hydration maltose) by the used time, and tissue culture blade scrapes mycelia, Acutely be vortexed concussion release spore, with filtered through gauze, blood counting chamber adjust under the microscope spore concentration to 2 × 105spores/ml.Before inoculation, the organosilicon for being added 2/10000ths extends agent as surface, is equably sprayed suspension with watering can It applies on blade, using inoculation medium as control.After inoculation, plant is placed in the space of 95% or more humidity, and temperature is general Blade incidence is observed in control after 22 DEG C or so, 2 days.

Sick grade index is counted according to blade incidence, as shown in Fig. 2, the incidence of control is obviously tighter compared with mutant Weight；Incidence of leaf is taken to carry out Trypan Blue, as shown in figure 3, the spot number of control is significantly more than mutant.

The statistical of sick grade index are as follows: be classified the tomato leaf of morbidity, grade scale are as follows: 0 grade of expression is not sent out Disease, the visible a small number of scabs of 1 grade of expression blade lower epidermis, 2 grades of expression blade lower epidermis local dense scabs, under 3 grades of expression blades The intensive scab of epidermis multiple location, the visible scab distribution of 4 grades of expression blade lower epidermis full leafs.0-4 rank distinguishes assignment 0-4 points.Root Incidence according to each blade of each plant is classified by classification, and each processing at least counts 50 tomato leafs.The state of an illness The calculation formula of index is as follows:

Disease index DI=∑ (numbers of blade at different levels × corresponding series) × 100%/(total leaf number × highest morbidity series)

To sum up, tomato pub41#1 gene mutation body material can significantly improve its resistance to graw mold of tomato.

4 tomato pub41#1 gene editing mutant of embodiment influences Resistance to bacterial wilt

Bacterium solution is in TTC screening and culturing medium (Peptone 10g/L；Casamino Acid 1g/L；Glucose 5g/L； Agar 15g/L；After autoclave sterilization, be added 1%TTC 5ml/L) on draw plate, be put in after being cultivated 2 days in 28 DEG C of incubators, Bacterium solution pathogenicity is detected, pathogenic strong bacterial strain, bacteria colony white, intermediate pink is in irregular shape, band viscosity.Bacterium solution is expanding Breeding culture medium (MgSO47H2O 0.3g/L；K2HPO4 2.0g/L；Yeast extract 4.0g/L；Casamino Acid 8.0g/L；Sucrose 10.0g/L；Agar 15.0g/L) on draw plate, be put in after being cultivated 2 days in 28 DEG C of incubators, with connecing collarium Picking band viscosity has the milky bacterium solution of mobility (beef extract 3g/L, peptone 10g/L, NaCl in NA culture medium 10g/L), cultivate 48h in 28 DEG C, the shaking table of 200rpm/min, 4 DEG C, 4000rpm/min be centrifuged 10min, with sterile water weight It is outstanding, bacterium solution OD600=1.0-1.5, pouring root inoculation are adjusted, every plant of 40ml bacterium solution is put in 28 DEG C of moisturizing environment, sees after 7 days Examine plant incidence.

Tomato pub41#1 gene editing mutant material and control carry out phenotype such as Fig. 4 after bacterial wilt germ is inoculated with 7 days It is shown.According to blade incidence, sick grade index is counted, as shown in figure 5, the incidence of control is obviously tighter compared with mutant Weight.

Sick grade index statistical are as follows: plant disease symptom is divided into five grades, grade scale by light and heavy degree are as follows: 0 grade, blade does not have illness normally；1 grade, the blade of 1%-25% is wilted；2 grades, the blade of 26%-50% withers It is listless；3 grades, the blade of 51%-75% is wilted；4 grades, the blade of 76%-100% is wilted.Each experiment at least repeats 3 It is secondary.The calculation formula of disease index is as follows:

Disease index DI=∑ (diseased plant numbers at different levels × corresponding series) × 100%/(investigation total strain number × highest morbidity grade Number)

To sum up, tomato pub41#1 gene mutation body material can significantly improve its resistance to bacterial wilt of tomato.

Embodiment 5PUB41 acts on tomato protein kinase ubiquitination and identifies

External ubiquitination reaction occurs in 30 μ l systems, comprising: in ubiquitination buffer (0.1M Tris-Cl, PH= 7.5,25mM MgCl2,2.5mM DTT, 10mM ATP) in be added 0.25 μ g E1 (Boston Biochem), 0.5 μ g E2 (Boston Biochem), 1.25 μ g HA-tagged ubiquitin (Boston Biochem), 1 μ g GST-PUB41 purifying Albumen is as E3 ubiquitin ligase.1 μ g MBP- tomato protein kinase purifying protein is as substrate protein.

2 × SDS-PAGE sample-loading buffer is added in 30 DEG C of incubation 2h in reaction, and 95 DEG C of metal baths boil 5min.Sample carries out Polyacrylamide gel electrophoresis, and hybridized with HA, GST and MBP antibody.As a result as shown in Figure 6.

The result shows that when in system there are when E1, E2, ubiquitin molecule, PUB41 and tomato protein kinase, tomato albumen Kinases can be by PUB41 ubiquitination.

Sequence table

<110>Zhejiang University

<120>PUB41 gene is improving the application in graw mold of tomato and Resistance to bacterial wilt as negative regulatory factor

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Lys Ala Ile Asp Tyr Ile Ser Ala Thr Lys Ala Ser Met Asp Ala Val

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Lys Met Thr Ala Glu Phe Leu Val Gly Lys Leu Ala Thr Gly Ser Pro

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Asp Ile Gln Arg Lys Ala Ala Tyr Glu Leu Arg Leu Leu Ala Lys Ser

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Gly Met Asp Asn Arg Arg Ile Ile Ala Glu Ser Gly Ala Ile Pro Phe

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Leu Thr Thr Leu Leu Asp Ser Arg Asp Pro Arg Ile Gln Glu Asn Ala

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Val Thr Ala Leu Leu Asn Leu Ser Ile His Glu Asn Asn Lys Ile Leu

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Ile Met Ser Ala Gly Ala Ile Asp Ser Leu Ile Arg Val Leu Gln Ser

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Gly Gln Thr Met Glu Ala Arg Gly Asn Ala Ala Ala Ala Ile Phe Ser

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Ala Ile Pro Ala Leu Val Gly Leu Leu Lys Asp Gly Thr Thr Ala Gly

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Asn Arg Gln Cys Ile Val Leu Ala Gly Ala Val Pro Leu Leu Ile Asp

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Leu Ser Leu Ile Leu Gly Cys Asn Glu Gly Leu Gln Ala Leu Arg Lys

465 470 475 480

Ser Arg Ile Leu Val Pro Leu Leu Val Asp Leu Leu Arg Phe Gly Ser

485 490 495

Ser Lys Gly Lys Asp His Ser Ile Thr Leu Leu Leu Gly Leu Cys Lys

500 505 510

Asp Thr Gly Glu Glu Ile Val Gly Arg Leu Leu Met Asn Pro Arg Ser

515 520 525

Ile Pro Ser Leu Gln Ser Leu Ser Ala Asp Gly Ser Leu Arg Ala Arg

530 535 540

Arg Lys Ala Asp Ala Leu Leu Arg Leu Leu Asn Arg Cys Cys Phe Gln

545 550 555 560

<210> 3

<211> 20

<212> DNA

<213>tomato (Solanum lycopersicum L.)

<400> 3

cacttgacat tttacctctg 20

<210> 4

<211> 24

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 4

attgcacttg acattttacc tctg 24

<210> 5

<211> 25

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 5

aaaccagagg taaaatgtca agtgc 25

<210> 6

<211> 22

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 6

ctacttatcg tcatcgtctt tg 22

<210> 7

<211> 20

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 7

gctctttgtg gaacctcatg 20

<210> 8

<211> 20

<212> DNA

<213>artificial sequence (Artificial sequence)

<400> 8

cctatcacag ttagtagcca 20

Claims (7)

1.PUB41 gene is improving the application in graw mold of tomato resistance as negative regulatory factor, which is characterized in that described The nucleotide sequence of the protein-coding region of PUB41 gene is as shown in SEQ ID NO.1.

Application of the albumen of 2.PUB41 gene coding as negative regulatory factor in raising graw mold of tomato resistance, feature exist In the amino acid sequence of the albumen is as shown in SEQ ID NO.2.

3. application as claimed in claim 2, which is characterized in that the albumen by being specifically bound with tomato protein kinase, By tomato protein kinase ubiquitination, and then negative regulation tomato is to the resistance of gray mold.

4.PUB41 gene is improving the application in bacterial wilt of tomato resistance as negative regulatory factor, which is characterized in that described The nucleotide sequence of the protein-coding region of PUB41 gene is as shown in SEQ ID NO.1.

5. a kind of method for the tomato for cultivating botrytis resistant and bacterial wilt characterized by comprising

(1) target fragments containing PAM structure are chosen in the protein-coding region of tomato PUB41 gene, with target fragments PAM structure Preceding 20bp is foundation, carries out design of primers, constructs CRIPR/Cas9 carrier；

(2) the Agrobacterium genetic engineering bacterium of the CRIPR/Cas9 carrier of (1) containing step is constructed；

(3) step (2) genetic engineering bacterium is converted into tomato cotyledon, obtain without external source Cas9 albumen and stablizes the pure of heredity Close mutant strain.

6. method as claimed in claim 5, which is characterized in that the nucleotide of 20 bases before the target fragments PAM structure Sequence is as shown in SEQ ID NO.3.

7. method as claimed in claim 5, which is characterized in that construct the nucleotides sequence of the primer of the CRIPR/Cas9 carrier Column are as shown in SEQ ID NO.4 and SEQ ID NO.5.