CN103103208B - Haynaldia villosa disulfide isomerase gene and application thereof - Google Patents
Haynaldia villosa disulfide isomerase gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of genetic engineering and discloses a haynaldia villosa disulfide isomerase gene and an application thereof. The cDNA (complementary deoxyribonucleic acid) sequence of Hv-PDI (Hantaan Virus-Protein Disulfide Isomerase) is SEQ ID NO. 1, and the encoded amino acid sequence is SEQ ID NO. 2. The gene derives from diploid haynaldia villosa (haynaldia villosa VV, 2n=14), is induced by powdery mildew in powdery mildew-resistant diploid haynaldia villosa, and is greatly expressed. Through transient expression, the gene is transformed to an infected wheat variety Yangmai 158, a result shows that excessive expression of Hv-PDI is capable of reducing the haustorium index of Yangmai 158. Therefore, the Hv-PDI is expectedly used for genetic engineering breeding, and the resistance of wheat to powdery mildew is hopefully improved when the Hv-PDI is introduced to wheat varieties susceptible to powdery mildew.
Description
Technical field
The invention belongs to genetically engineered field, disclose a cluster hair wheat disulphide isomerase gene and application thereof.
Background technology
The wheat powdery mildew being caused by obligatory parasitism fungi wheat powdery mildew (Blumeria graminis DC f.sp.tritici) is China and in many national Wheat Production, endangers one of disease being on the rise in the world.From a leaf phase to aging, plant can be infected by powdery mildew.Early stage infecting can make minimizings of tillering, and infecting of upper blade and fringe portion late period can seriously reduce seed product (20%-33%).
Although Powdery Mildew can be used bactericidal agent for preventing and treating, but must increase human and material resources drops into, but also can cause the ecological problems such as environmental pollution, therefore cultivation and popularization disease-resistant variety have been acknowledged as control wheat powdery mildew economy, safety and effective approach the most, and the research of disease-resistant genetic background and disease-resistant mechanism is the important evidence of formulation resistant breeding strategy.Define its disease-resistant mechanism, clone's disease-resistant related gene, for control wheat diseases, carry out breeding for disease resistance improvement and have most important theories directive significance.
Disease-resistant wheat molecular mechanism is the hot subject of current wheat powdery mildew research.Interaction between powdery mildew pathogenic bacteria and host's wheat is very complicated.Expression conditions in analysis of pathogenic bacteria-host Interaction, contributes to excavate disease-resistant gene and finds disease-resistant path, and further illustrating disease-resistant molecular mechanism.By structure, be subject to cDNA library, the SSH library of Powdery Mildew abduction delivering, utilize two-dimensional electrophoresis connexus spectral technology, chip technology etc., filtered out gene or albumen that may be relevant to powder mildew resistance, comprise various pathogenesis-related proteins, serine-threonine kinase, abc transport albumen etc., also found the signal paths such as salicylate pathway, hydrogen peroxide approach and powder mildew resistance pass.
Dasypyrum villosum is the wheat of high mildew-resistance edge species far away, its disease-resistant gene Pm21 is positioned on 6V, this laboratory early-stage Study shows that Hv-CMPG is relevant to the Powdery Mildew of cluster hair wheat, and this gene is an E3 ligase enzyme, participates in ubiquitination process degraded combining target substrate protein.Research is study hotspot in recent years as Binding Capacity albumen and the signal transduction path of this class of Hv-CMPG E3 ligase enzyme.
Summary of the invention
The object of the invention is the above-mentioned defect for prior art, a kind of disulphide isomerase gene Hv-PDI is provided.
Another object of the present invention is to provide the expression vector of this gene.
Another object of the present invention is to provide the application of this gene and expression vector.
Object of the present invention can be achieved through the following technical solutions:
Disulphide isomerase gene Hv-PDI, from Dasypyrum villosum (Haynaldia villosa, VV, 2n=14), can do mutually with powder mildew resistance genes involved Hv-CMPG, and its nucleotides sequence is classified SEQ ID NO.1 as.
The protein Hv-PDI of this receptor kinase gene coded protein, its aminoacid sequence is SEQ ID NO.2.
The expression vector that contains described disulphide isomerase gene Hv-PDI.
The described expression vector that contains disulphide isomerase gene Hv-PDI claimed in claim 1 preferably with pBI220 for the carrier that sets out, HvPDI gene claimed in claim 1 is inserted to gained between the BamH/ of pBI220 and Kpnl restriction enzyme site.
The application of described disulphide isomerase gene Hv-PDI in building powdery-mildew-resistance wheat kind.
The application of the described expression vector containing disulphide isomerase gene Hv-PDI in building powdery-mildew-resistance wheat kind.
The present invention clones first and has obtained one and the mutual disulphide isomerase gene Hv-PDI doing of powder mildew resistance genes involved HvCMPG and coded protein Hv-PDI thereof from cluster hair wheat, is reported first in cluster hair wheat.Be inserted into expression vector pBI220, the Overexpression vector of this gene obtaining imports in susceptible wheat breed, can improve the resistance of sense Powdery Mildew wheat breed to Powdery Mildew.Hv-PDI, for genetic engineering breeding, is imported in susceptible Powdery Mildew wheat breed, can improve the powder mildew resistance of wheat.
Beneficial effect:
The present invention utilizes yeast-two hybrid technique, using Hv-CMPG as bait, utilize polyoxyethylene glycol-Lithium Acetate conversion method, screening is subject to the cluster hair wheat Yeast two-hybrid cDNA library of powdery mildew induction, obtain an interacting genes Hv-PDI of Hv-CMPG, this gene is a disulphide isomerase, is subject to powdery mildew induction up-regulated expression in cluster hair wheat blade; Be inserted into expression vector pBI220, the Overexpression vector that obtains this gene imports in susceptible wheat breed, can improve the resistance of sense wheat powdery mildew kind to Powdery Mildew.Therefore, Hv-PDI can be used for genetic engineering breeding, is particularly applied to the wheat breed that genetic engineering means is cultivated mildew-resistance.
Accompanying drawing explanation
Fig. 1 utilizes the interacting genes Hv-PDI of yeast two-hybrid clone Hv-CMPG
Upper behavior: SD-LT substratum
Lower behavior: SD-HLT substratum; Be respectively from left to right pGADT7+pGBKT7; PGBKT7:CMPG+pGADT7;
pGADT7：PDI+pGBKT7；pGBKT7：CMPG+pGADT7：PDI。
QRT-PCR in the blade of Fig. 2 Hv-PDI after the induction of Dasypyrum villosum powdery mildew analyzes
Up: Hv-PDI is subject to the expression of the different time of powdery mildew induction at cluster hair wheat blade;
Middle row: house-keeping gene Tubulin is subject to the expression of the different time of powdery mildew induction at cluster hair wheat blade;
Descending: 0h, 30min, 45min, 1h, 2h, 6h, 12h, 24h, 48h, 72h represent that respectively cluster hair wheat blade is subject to the different time sections of powdery mildew induction
Fig. 3 Hv-PDI Overexpression vector builds.
The epidermic cell of the expression gus gene that Fig. 4 and powdery mildew are done mutually
A: powdery mildew does not form haustorium after invading the epidermic cell of expressing GUS;
B: powdery mildew forms haustorium after invading the epidermic cell of expressing GUS
Co: conidium; Pp: invade nail; Ha: haustorium; Hy: mycelia
Fig. 5 utilizes the effect of unicellular Transient Expression technical study Hv-PDI in mildew-resistance
Embodiment
Embodiment 1 utilizes yeast two-hybrid clone cluster hair wheat disulphide isomerase gene Hv-PDI
Dasypyrum villosum (reference: Qi Lili, Chen Peidu, etc., wheat powdery mildew new resistance source-gene Pm 21, Acta Agronomica Sinica, 1995,21 (3): 257-262) be the material of high mildew-resistance.Hv-CMPG be of cytogenetics institute of Agricultural University Of Nanjing clone be positioned at powder mildew resistance genes involved on Dasypyrum villosum 6V (Liu Yuan. cluster hair wheat Hv-CMPG gene clone and function initial analysis [D] thereof. Nanjing. Agricultural University Of Nanjing, 2007), take Hv-CMPG as bait, with polyoxyethylene glycol-Lithium Acetate conversion method screening Yeast two-hybrid cDNA library, obtain the interacting genes (accompanying drawing 1) of a Hv-CMPG.To this interacting genes order-checking, having obtained size is the sequence of 1615bp, and sequence is as shown in SEQ ID NO.1.By the ORF finder in NCBI website, search for the open reading frame of this acquisition sequence, find the gene that it comprises a total length ORF, 5 '-UTR(non-translational region wherein) 81bp, 3 '-UTR211bp, ORF(open reading frame) 1323bp, 440 amino acid of encoding, sequence, as shown in SEQ ID NO.2, is Hv-PDI by this unnamed gene.
Embodiment 2Hv-PDI gene is subject to the expression characteristic of powdery mildew induction
The cluster hair wheat seed (reference: Qi Lili of mildew-resistance, Chen Peidu, Deng, wheat powdery mildew new resistance source-gene Pm 21, thing journal, 1995,21 (3): 257-262) be sowed in culture dish and germinate, after showing money or valuables one carries unintentionally, be transplanted to basin alms bowl (with cylindric transparent plastic sheet isolation, top is sealed with filter paper, forms the environment without powdery mildew) around.Treat tri-leaf period, the Nanjing fresh spore of mixing powdery mildew of cultivating on susceptible variety Soviet Union wheat No. three is shaken off on the seedling at cluster hair wheat gently.Cluster hair wheat blade after inoculation powdery mildew is kept at 16 ℃.After inoculation, 0h, 30min, 45min, 1h, 2h, 6h, 12h, 24h, 48h, 72h sampling, be placed in-70 ℃ of refrigerators and save backup.With TRIZOL(Invitrogen) extract the RNA of the cluster hair wheat blade be subject to powdery mildew induction, utilize AMV enzyme (Takara) to synthesize reverse transcription the first chain, obtain reverse transcription product.
Apply the special primer P1(CAAGTGGCAAAACCTCTGGT(SEQ I D NO.3 of energy specific amplified Hv-PDI)) and P2(TACAAAGCAAATGGCAGCAG(SEQ ID NO.4)), this gene is carried out to sxemiquantitative PCR(sQ-RT-PCR in being subject to powdery mildew induction sample) analyze.PCR reaction is in the upper amplification of PCR instrument (PTC-200, Bio-Rad, USA).In 10 μ l PCR reaction systems, contain 1 μ l1 * Buffer, 1.5mmol/L MgCl2,200mmol/L dNTP, 0.2nmol/ μ l primer P1 and P2,0.5UTaqDNA polysaccharase (rTaq TakaRa), the reverse transcription product 1 μ l of above-mentioned acquisition.Amplification is: 95 ℃ of 3min, then 95 ℃ of 30s, 56 ℃ of 45s, 72 ℃ of 1min, totally 26 circulations.After reaction finishes, reaction product adds after 2 μ l loading buffer the agarose gel electrophoresis with 1%.Result shows, and house-keeping gene (Tubulin) compares, and in cluster hair wheat blade, Hv-PDI is subject to powdery mildew induction up-regulated expression, and after 24h, expression level reaches peak value, starts afterwards to lower.The result of sQ-RT-PCR shows, Hv-PDI may relevant to powder mildew resistance (accompanying drawing 2).
Embodiment 3Hv-PDI sense expression vector builds
Utilizing above-mentioned cluster hair wheat cDNA after powdery mildew induction is template, primer pair P3(CGGGATCCATGCGTCCGGCCATCC(SEQ ID NO.5 with the Hv-PDI gene protein coding region of can increasing)) and P4(GGGGTACCTCACAACTCGTCGTTGGGC(SEQ ID NO.6)) carry out pcr amplification, reclaiming size is 1323bp amplified fragments.Amplified fragments is inserted in pMD18-T (Takara), through order-checking after and former sequence alignment, for Hv-PDI gene, with BamHI and Kpnl double digestion, amplification target segment is inserted into carrier pBI220(Jefferson RA, Kavanagh TA, Bevan MW.GUS fusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J.1987, between the multiple clone site BamHI and Kpnl below of 35S promoter 6:3901-3907.).Thus target gene Hv-PDI is cloned into the downstream of strong promoter 35S, after order-checking, and former sequence alignment is without point mutation, obtains expression vector pBI220:PDI(accompanying drawing 3).
Embodiment 4 utilizes Transient Expression method that Hv-PDI gene is proceeded to wheat leaf blade
Powdery mildew can form haustorium in about about 24 hours after infecting sense Powdery Mildew wheat leaf blade in blade epidermis, and haustorium is to absorb host's nutrient for the important structure of powdery mildew mycelial growth.If powdery mildew can form haustorium after infecting and be commonly considered as host-pathogenic bacteria compatible reaction has occurred in blade table chrotoplast, what blade produced is susceptible reaction; If can not form haustorium in blade table chrotoplast, be commonly considered as host-pathogenic bacteria non-compatible reaction has occurred, blade has produced disease resistance response.Haustorium index, the cell proportion that has haustorium to form in the cell of doing mutually with pathogenic bacteria, is an important indicator weighing host resistance.
Transient Expression method is a kind of method (reference: Schweizer of reliable and Rapid identification gene function, Pokorny et al.A Transient Assay System for the Functional Assessment of Defense-Related Genes in Wheat.Molecular Plant-Microbe Interactions.1999, 12:647-654.), plasmid DNA can be wrapping to metal particle skin, by particle gun, metal particle is bombarded to the epidermic cell of wheat leaf blade, the goal gene carrying on carrier overexpression in blade cell.
This research is building up to pBI220:PDI in overexpression carrier by Hv-PDI, by Transient Expression method by pBI220:PDI and gus gene place carrier pAHC25(Christensen A H, Quail P H.Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants.Transgenic Research, 1996, 5:213-218.) carry out cotransformation, cell with gus gene mark Hv-PDI genetic expression, by relatively bombarding the powdery mildew haustorium index and the powdery mildew haustorium index that does not bombard Hv-PDI cell of Hv-PDI cell, whether hard objectives gene has powdery mildew disease-resistant function.Concrete operation step is as follows:
The program of carrier DNA and metal particle parcel: 1, prepare tungsten powder: (1) takes the tungsten powder of 30mg in 1.5ml eppendorf pipe, adds 1ml70% alcohol, and standing 15min after vortex 3-5min, precipitates tungsten powder completely.(2) after the centrifugal 1min of 12000rpm, abandon supernatant.(3) in precipitation, add 1ml ddH
2o water, after vortex mixes, the centrifugal supernatant (in triplicate) of abandoning of 12000rpm.(4) in precipitation, add 500 μ l glycerine (V/V, 50%) vortexs to mix, with standby.2, parcel bullet: (1) draws the uniform tungsten powder of 5 μ l vortex in the eppendorf of 1.5ml pipe, adds 5 μ l plasmid DNA (concentration of 0.2 μ g/ μ l), and vortex limit, limit drips 50 μ l CaCl in eppendorf pipe
2(2.5M), then add 20 μ l spermidine (0.1M now joins first and uses), vortex 3min.(2) centrifugal 2s after standing 1min, abandons supernatant, in precipitation, adds 140 μ l alcohol (V/V, 70%), abundant vortex, and centrifugal 2s, abandons supernatant.(2) add 140 μ l raw spirits, abundant vortex, centrifugal 2s, abandons supernatant.In precipitation, add 15 μ l raw spirits, fully vortex, prepares against and uses.
Particle gun bombardment program is as follows:
Cut the wheat seedlings blade end that is about 6cm, parallel being attached on slide glass, every slide pastes 6 blade left and right.Particle gun is used PDS1000/He system, adopts the split diaphragm of 1350psi, and vacuum tightness is 28inHg.After bombardment, blade is placed in the porcelain dish that is lined with wetting filter paper, the preservative film of cover to be with holes, moisturizing is also ventilative, after 18-20 ℃ of renewal cultivation 4h, high-density inoculation powdery mildew conidium.After inoculation 48h, with GUS dye liquor, [formula is: 0.1M Na
2hPO
4/ NaH
2pO
4damping fluid (pH7.0), containing 10mM EDTA, the 5mM Tripotassium iron hexacyanide and yellow prussiate of potash, 0.1mg/ml X-Gluc, 0.1%Triton X-100,20% methyl alcohol] vacuum infiltration 10min, 37 ℃ of dyeing 12h, then with 70% alcohol, decolour 2 days until after blade bleaching, use Xylene Brilliant Cyanine G (W/V, 0.6%) to powdery mildew spore staining.
While implementing the conversion of gus gene list, by the plasmid DNA that contains gus gene expression vector pAHC25 and tungsten powder parcel; When implementing Hv-PDI and gus gene cotransformation, the plasmid DNA that contains Hv-PDI expression vector pBI220:PDI is mixed to parcel tungsten powder with the plasmid DNA that contains gus gene expression vector pAHC25 in the volumetric molar concentration ratio of 1: 1.When gus gene and Hv-PDI gene carry out cotransformation, the dyeing that marker gene GUS proceeds to presents blue cell and is the cell that Hv-PDI proceeds to.
The disease-resistant functional analysis of embodiment 5Hv-PDI
In the cell of expressing at GUS, finger-like haustorium can be dyed blueness by GUS staining fluid, under the microscope easily identification.After gus gene transformant, in the GUS express cell of doing mutually by statistics and powdery mildew, the shared ratio (%) of cell that haustorium forms, be " haustorium index " (reference: Schweizer, Pokorny et al.A Transient Assay System for the Functional Assessment of Defense-Related Genes in Wheat Molecular Plant-Microbe Interactions.1999,12:647-654).Haustorium index is less, and the cell proportion that shows to form haustorium is lower, and the disease resistance of blade is stronger.This research and utilization " haustorium index " is as the measurement index of disease-resistant power.
When single conversion gus gene, the haustorium index in sense Powdery Mildew wheat breed Yangmai No.158 is that 63.28%(has added up 897 cells).
After gus gene and Hv-PDI corotation allelopathic Powdery Mildew wheat breed Yangmai No.158, added up gus gene expression (being that Hv-PDI expresses) and had the powdery mildew haustorium index of the cell of work mutually, result shows after Hv-PDI proceeds to, and the haustorium index of Yangmai No.158 drops to 37.55%(from 63.28% and added up 1055 cells).This presentation of results, Hv-PDI can reduce haustorium index significantly, and its Transient Expression can improve the resistance to powdery mildew.
Claims (2)
1. nucleotides sequence is classified the disulphide isomerase gene of SEQ ID NO.1 as
hv-PDIapplication in cultivating powdery-mildew-resistance wheat kind.
2. containing nucleotides sequence, classify the disulphide isomerase gene of SEQ ID NO.1 as
hv-PDIthe application of expression vector in cultivating powdery-mildew-resistance wheat kind.
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| CN103436516B (en) * | 2013-08-23 | 2015-04-22 | 华南理工大学 | Wheat protein disulfide isomerase produced by utilizing escherichia coli prokaryotic expression system, as well as method and application of wheat protein disulfide isomerase |
| CN105821055B (en) * | 2015-01-04 | 2019-08-13 | 王秀娥 | One haynaldia villosa Pleurotus Ostreatus receptor kinase gene and its expression vector and application |
| CN110229826B (en) * | 2019-06-18 | 2021-10-19 | 南京农业大学 | Haynaldia villosa CEBiP1-V gene and protein coded by same and application thereof |
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