CN103305486B - Application of wheat TaCPK2 protein in plant disease-resistant breeding - Google Patents
Application of wheat TaCPK2 protein in plant disease-resistant breeding Download PDFInfo
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Abstract
The invention discloses application of a wheat TaCPK2 protein in plant disease-resistant breeding. The invention has the following advantages: a wheat TaCPK2 gene provided by the invention belongs to genes of the CDPK gene family participating in plant defense against causes of diseases, inhibition of expression of the TaCPK2 gene in wheat can obviously influence resistance of wheat to powdery mildew, and overexpression of the TaCPK2 gene in paddy rice can obviously improve resistance of transgenic rice to bacterial leaf blight; and the TaCPK2 gene can be used for overcoming the problem of decrease of output of plants caused by fungous or bacterial diseases like powdery mildew and bacterial leaf blight.
Description
Technical field
The present invention relates to genetically engineered field, particularly relate to the application of a calcium-dependent protein kinase gene in disease-resistant engineering.
Background technology
In recent years, due to a large amount of uses of sterilant, although there is not the situation of crop pest big area outburst, yet too much depend on agricultural chemicals, not only increased production cost, but also caused big area environmental pollution, serious threat Agro-ecology safety.Adopt animal nutrition, cloning crop disease-resistant gene and being applied to crop disease-resistant genetic breeding is to control popular most economical, the safe and effective method of crop pest.Yet, the mutual specialization resistance of kind to microspecies that show as due to disease-resistant gene and nontoxic gene, disease resistance of plant is often overcome because of the variation of pathogenic bacterium colony physiological strain, and some disease-resistant gene is not also cloned into, and its disease resistance is just because the variation of germ Population Toxicity is lost.As can be seen here, utilizing disease-resistant gene to cultivate disease-resistant variety is also constantly being faced with new challenges.People then start screen wide spectrum, lasting disease-resistant important genes involved and cultivate the crop varieties with Durable resistance.
Calcium-dependent protein kinase (Calcium-dependent protein kinase, CDPK) is the peculiar calcium ion (Ca of plant and protozoon
2+) susceptor.The stimulations such as abiotic stress, cause of disease, hormone and light all can cause plant endogenous Ca
2+concentration change (Knight and Knight, 2001; Sanders, 2002), CDPK can identify Ca
2+change in concentration, changes downstream egg white matter (as transcription factor) phosphorylation state, affects gene expression pattern (Sanders et al., 1999).CDPK albumen comprises 4 structural domains, i.e. N-end variable region (N), protein kinase structural domain (kinase, K), Zi Yi district (autoinhibitory region, A) and class calmodulin structural domain (CaM-like) (Cheng et al., 2002; Hrabak et al., 2003).CaM-like structural domain comprises can be in conjunction with Ca
2+eF hands functional domain.Work as Ca
2+the CaM-like structural domain that is attached to CDPK, Zi Yi district is disengaged the inhibition in kinases territory, and CDPK is activated.CDPK is playing the part of the role of crucial regulon in many barss of plant bang path.
As the early signal factor, CDPK, in signaling path upstream position, plays an important role (Cheng et al., 2002) in pathogenic defence.Tobacco NtCPK2 is found to be the component (Romeis et al., 2001) of the signaling path of R gene mediated.In barley, transient expression HvCDPK4, induction spreads with the necrocytosis of the similar tobacco mesophyll of anaphylaxis, is unfavorable for the intrusion of fungi; Transient expression HvCDPK3 is conducive to the intrusion (Freymark et al., 2007) of fungi.This enters HvCDPK3 in barley and HvCDPK4 in the process of host cell at control white powder morbidity commitment gene, plays antagonistic action.OsCPK13 in paddy rice (OsCDPK7) is at salt, and (Saijo et al., 2000 play an important role in the adverse circumstance environment such as arid and low temperature; Komatsu et al., 2007), it is crossed and expressed in jowar, but show disease-resistant correlated character (Mall et al., 2011).In potato, StCDPK5 induces the phosphorylation of StRBOHB and regulates oxidative burst (Kobayashi et al., 2007), may participate in the defense response of plant to cause of disease.
Although wheat CDPK family studies have reported that (Li et al., 2008) at present, however the not clearly explaination of the biological function of relevant TaCPK2.
Summary of the invention
In order to address the above problem, the object of the present invention is to provide the application of grow wheat TaCPK2 albumen in Resistant breeding.
To achieve these goals, the invention provides a grow wheat TaCPK2 albumen, its aminoacid sequence is as shown in SEQ ID No.2.
The invention provides the application of wheat TaCPK2 albumen in Resistant breeding process.
Described disease is preferably Powdery Mildew or bacterial leaf-blight; Described plant optimization is wheat or paddy rice.
The application of wheat TaCPK2 albumen provided by the invention in regulating plant is disease-resistant.Described application refers to by crossing expression wheat TaCPK2 gene, regulates SA, the expression variation of JA path coherence gene, thus make plant produce disease resistance.
Technique scheme tool has the following advantages:
Wheat TaCPK2 gene of the present invention, it belongs to the gene of CDPK gene family involved in plant to cause of disease defence, the expression that suppresses TaCPK2 gene in wheat can obviously affect wheat to powder mildew resistance, and cross expression TaCPK2 gene in paddy rice, can obviously increase transgenic paddy rice to bacterial leaf spot resistance.Can be used for solving plant in the problems such as output minimizing that cause due to the fungies such as Powdery Mildew, bacterial leaf-blight or bacterial disease.
Accompanying drawing explanation
Fig. 1 is clone's intermediate carrier pEASY-T1 Simple of the embodiment of the present invention 1 and embodiment 3 wheat TaCPK2 genes;
Fig. 2 is the Phenotypic Observation that the disease resistance after the wheat calcium-dependent protein kinase TaCPK2 gene VIGS of the embodiment of the present invention 4 reduces;
Fig. 3 is the plant expression vector pCUbi1390 of the embodiment of the present invention 5;
Fig. 4 is that the wheat calcium-dependent protein kinase gene TaCPK2 of the embodiment of the present invention 6 increases the resistance of paddy rice (seedling stage) to bacterial leaf spot;
Fig. 5 is that the wheat calcium-dependent protein kinase gene TaCPK2 of the embodiment of the present invention 7 increases the resistance of paddy rice (strain phase) to bacterial leaf spot;
Fig. 6 is that the quantitative statistics result of the embodiment of the present invention 8 shows that wheat calcium-dependent protein kinase gene TaCPK2 increases the resistance of Rice Seedlings to bacterial leaf spot;
Fig. 7 is that the embodiment of the present invention 9 is crossed RT-PCR positive identification and the native gene thereof of expressing rice plant and expressed constant;
Fig. 8 is that the wheat calcium-dependent protein kinase TaCPK2 gene overexpression paddy rice of the embodiment of the present invention 10 does not connect the species test result under bacterium condition.
Wherein, empty carrier (empty vector is called for short pC) is contrast, and 1,2,3 is that 3 paddy rice cross expression strain.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to protection scope of the present invention.
The biomaterial Chinese spring relating in the present invention, durum wheat DR147, wheat Am6, Beijing 837 are public kind.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
The clone of embodiment 1 wheat calcium-dependent protein kinase TaCPK2 full length gene
Use is with the primer of restriction enzyme site, forward primer GCAT
aTGGGCAACGCATGCGGCGGT (as shown in SEQ ID No.4) and reverse primer GCAT
cTAGATTGCACCAGGTGCGTC (as shown in SEQ ID No.5) clones the coding region sequence of TaCPK2 gene from the cDNA of Chinese spring (Triticum aestivum L.) leaf;
PCR program: 94 ℃, 5 minutes; 94 ℃, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 90 seconds; Repeat 35 times; 72 ℃, 10 minutes.
PCR system: 2 * EasyTaq PCR SuperMix (Quan Shi King Company), 25 μ l;
Forward primer (10 μ M) 2 μ l;
Reverse primer (10 μ M) 2 μ l;
DNA profiling 5 μ l;
Distilled water is supplied 50 μ l.
PCR product the pEASY-T1 Simple Cloning Kit cloning process clone who provides according to Beijing Quanshijin Biotechnology Co., Ltd after glue recovery purifying is provided and is connected to (Fig. 1) on pEASY-T1Simple carrier, connect product and transform bacillus coli DH 5 alpha, and expand therein numerous, positive colony obtains TaCPK2 through order-checking screening, and its cDNA sequence is as shown in SEQ ID NO.1; By the aminoacid sequence of the protein of its coding as shown in SEQ ID NO.2.
Embodiment 2: the structural analysis of wheat calcium-dependent protein kinase gene TaCPK2 proteins encoded
The structural analysis of wheat calcium-dependent protein kinase gene TaCPK2 proteins encoded shows, by the albumen of this coded by said gene, with the CDPK protein structure domain in other plant, equally has conservative enzyme live site and structural domain.Phylogenetic analysis demonstration, the highest with the HvCDPK4 protein similar in barley by the albumen of wheat TaCPK2 coded by said gene, be 95.9%.And spreading of HvCDPK4 induction mesophyll cell death is unfavorable for the intrusion of fungi, the intrusion of powdery mildew is played to resistant function.Therefore, the HvCDPK4 gene in wheat TaCPK2 gene and barley has the similar function of resisting powdery mildew invasion.
Embodiment 3: VIGS carrier and the VIGS plant of wheat calcium-dependent protein kinase gene TaCPK2
Use is with the primer of restriction enzyme site, forward primer
tGTCCTTTGATGGGCAACGCA (as shown in SEQ ID No.6) and reverse primer
cCGCGTGGCCGTCCGTCTT (as shown in SEQ ID No.7) carries out pcr amplification, PCR program from the cDNA of Chinese spring (Triticum aestivum L.) leaf: 94 ℃, and 5 minutes; 94 ℃, 30 seconds; 56 ℃, 30 seconds; 72 ℃, 30 seconds; Repeat 35 times; 72 ℃, 10 minutes.
PCR system: 2 * EasyTaq PCR SuperMix (Quan Shi King Company), 25 μ l;
Forward primer (10 μ M) 2 μ l;
Reverse primer (10 μ M) 2 μ l;
DNA profiling 5 μ l;
Distilled water is supplied 50 μ l.
PCR product the pEASY-T1 Simple Cloning Kit cloning process clone who provides according to Beijing Quanshijin Biotechnology Co., Ltd after glue recovery purifying is provided and is connected on pEASY-T1 Simple carrier (Fig. 1), connect product and transform bacillus coli DH 5 alpha, and expand therein numerous, positive colony obtains the cDNA fragment of TaCPK2 through order-checking screening, its cDNA sequence is as shown in SEQID NO.3.Barly strip mosaic virus (BSMV) carrier is by BSMV-α, BSMV-β, and tri-components of BSMV-γ form (Holzberg et al., 2002; Zhao Dan etc., 2011).PEASY-T1Simple carrier and BSMV-γ carrier that above-mentioned order-checking is correct are cut with the NheI enzyme of NEB, reclaim TaCPK2 (190bp) gene fragment and BSMV-γ carrier segments that enzyme is cut, with T4DNA ligase enzyme (NEB), connect, the gene fragment of TaCPK2 is reversely connected to BSMV-γ carrier multiple clone site.Connection product is transformed in bacillus coli DH 5 alpha competence, and bacterium liquid PCR screening positive clone sequence verification, obtain the correct vector plasmid of sequence and be recombinant vectors BSMV-γ: TaCPK2.
Green fluorescent protein (Green Fluorescent Protein, GFP) derive from marine organisms jellyfish, fluorescence can be expressed and produce to its gene in allos tissue, the about 714bp of GFP cDNA open reading frame length, 238 amino-acid residues of encoding, the inner 65-67 position Serine-dehydrogenation of its peptide chain tyrosine-glycine by recirculation and oxidation form a chromophore because of, under long ultraviolet wavelength or blue light illumination, send green fluorescence.Green fluorescent protein is one of conventional reporter gene.In this research, why select green fluorescent protein in contrast, be because this gene in plant materials without any homologous gene, any gene of the BSMV carrier that therefore carries GFP in can reticent plant, can be used as gene and lowers the contrast of expressing.
In VIGS experiment, BSMV:GFP is usually used as negative control (Li et al., 2011; Cao et al., 2011).This vector construction process and BSMV-γ: TaCPK2 carrier is identical, the GFP gene fragment that is about to be cloned into and BSMV-γ for carrier segments T4DNA ligase enzyme (NEB) be connected, and by being transformed into competent escherichia coli cell, order-checking screening obtains correct vector plasmid and is BSMV-γ: GFP recombinant vectors.
The method steps that VIGS plant obtains:
1) extract BSMV-α, BSMV-β, the plasmid of BSMV-γ: TaCPK2 and BSMV-γ: GFP, BSMV-α, BSMV-γ: TaCPK2 and BSMV-γ: GFP cut with Mlu1 enzyme, and BSMV-β cuts with Spe1 enzyme, and 37 ℃, 8h.Linearization process, it is as follows that 50 μ l NEB enzymes are cut system:
Mlu1：
10 * NEB damping fluid: 5 μ l
Plasmid :≤2 μ g
Mlu1： 1μl
Sterilizing distilled water: to 50 μ l
Spe1：
10 * NEB damping fluid: 5 μ l
Plasmid :≤2 μ g
100×BSA： 0.5μl
Mlu1： 1μl
Sterilizing distilled water: to 50 μ l
2) ethanol precipitation
The good plasmid of 150 μ l linearizing, adds 350 μ l DEPC (Diethyl pyrocarbonate) H
2o, adds 500 μ l phenol chloroforms (phenol and chloroform volume ratio are 1: 1), the centrifugal 30min of 12000rpm.Get supernatant 450 μ l, (PH 5.2,3M), add 1ml cold (4 ℃) dehydrated alcohol, mix to add 45 μ l NaAc.-20 ℃ are spent the night.The centrifugal 30min of 12000rpm, outwells supernatant, and 1ml 70% ethanol repeats to wash twice, dries, and adds 30 μ l DEPC H
2o dissolves.Answer >=400ng/ μ l of concentration.
3) in-vitro transcription test kit is used the AmpliCap-Max that Beijing GBI company provides
tMt7and T3 High Yield Message Maker Kits (Epicentre, USA)
10 μ l reaction systems:
Template: 3 μ l
10 * transcribe damping fluid: 1 μ l
Cap/NTP premixed liquid: 4 μ l
100mM dithiothreitol (DTT): 1 μ l
T7 enzyme: 1 μ l
42℃，3h。
4) virus inoculation
(or BSMV-γ: GFP) transcript is respectively got 10 μ l and mixed, totally 30 μ l add the DEPC H of 3 times of volumes (90 μ l) for BSMV-α, BSMV-β, BSMV-γ: TaCPK2
2o, cumulative volume reaches 120 μ l, then the GKP buffer of the volume that doubles (120 μ l), makes the virus inoculation mixed solution of 240 μ l TaCPK2 and GFP.
Wheat lines for virus infection is selected mildew-resistance near isogenic line PmAm6/ Beijing 837*BC
5f
3(PmAm6/ Beijing 837*BC
5f
3preparation method: take tetraploid durum wheat (Triticum durum) DR147 as maternal, diploid aegilops tauschii (Aegilops taushii) Ae39 of take is male parent, synthetic hexaploid wheat Am6.Take Am6 as donor parents, take Beijing 837 as recurrent parent, from hybridization F1, screen mildew-resistance individual plant, backcross with Beijing 837, from each backcross progeny, screen disease-resistant individual plant.F from 5 generations that backcrossed
3in family, screen the disease-resistant individual plant that isozygotys, be PmAm6/Beijing837*BC
5f
3).When second leaf of wheat seedling flattens (14d), start inoculation.Wear firm Kaifeng rubber gloves, with the rifle head without RNA enzyme of processing, get 5-10 μ l inoculation liquid to forefinger tripe, fix on the other hand wheat seedling base portion, thumb and forefinger second leaf of wheat that rub back and forth gently for another hand.
After having inoculated, to wheat seedling, spray DEPC H
2o, preservative film covers, and moisturizing 24h (high humidity is conducive to Virus entry), opens afterwards.Temperature all can (temperature raises and to be unfavorable for Virus entry, and long very fast of blade) between 20-32 ℃.
After 6-8 days, the 3rd leaf produces viral plaque, is virus inoculation success, obtains the VIGS plant of BSMV:TaCPK2 and BSMV:GFP.
Embodiment 4:VIGS experimental results show that TaCPK2 gene participates in powder mildew resistance reaction
The BSMV:TaCPK2 obtaining in embodiment 3 is cultured to the 4th leaf with BSMV:GFP plant and flattens together with incubator, by there being the plant leaf of virus morbidity phenotype, cut, be placed on and on benzoglyoxaline substratum, inoculate powdery mildew E09 and cultivate one week, can obviously find out that in BSMV:TaCPK2 plant, TaCPK2 down regulation of gene expression causes infecting Powdery Mildew, blade surface has macroscopic white sorus, and contrast in BSMV:GFP plant because TaCPK2 genetic expression does not change, therefore mildew-resistance still, blade surface is clean.Reach a conclusion thus: TaCPK2 gene participates in powder mildew resistance reaction.As shown in Figure 2, Bj grows sorus after susceptible material inoculation powdery mildew, as susceptible contrast; MOCK is anti-white powder material inoculation GKP buffer, does not infect Powdery Mildew; BSMV:GFP is virus inoculation BSMV:GFP plant, does not infect Powdery Mildew; BSMV:TaCPK2 is virus inoculation BSMV:TaCPK2 plant, local infection Powdery Mildew, prove that TaCPK2 gene participates in powder mildew resistance reaction.
Embodiment 5: wheat calcium-dependent protein kinase gene TaCPK2 plant expression vector
The correct TaCPK2 of order-checking obtaining from embodiment 1, utilize two restriction enzyme sites of KpnI and SpeI, obtain being structured in the full length sequence TaCPK2 on pEASY-T1 Simple (Beijing Quanshijin Biotechnology Co., Ltd) carrier, by plant expression vector pCUbi1390 (Peng et al., 2009) (Fig. 3) carry out after same enzyme cuts, according to the using method of the T4 DNA ligase of NEB company, enzyme being cut to two fragments that obtain connects, and will connect product conversion bacillus coli DH 5 alpha according to the method in embodiment 3, expand therein numerous, positive colony obtains the over-express vector of TaCPK2 through order-checking.Adopt agrobacterium-mediated transformation that the over-express vector of the TaCPK2 obtaining is transformed in paddy rice, obtain transformed plant.Selection markers in plant is Totomycin.
Application Example 6: wheat calcium-dependent protein kinase gene TaCPK2 increases resistance---the seedling stage of paddy rice to bacterial leaf spot
The rice conversion plant obtaining from embodiment 5 is cultivated together with the while large Tanaka with its empty carrier (wild-type sky educates 131), until the strain phase.Fig. 4 shows wheat calcium-dependent protein kinase gene TaCPK2 rice transformation, causes Rice Seedlings to increase the resistance of bacterial leaf spot.As shown in Figure 4, empty carrier (empty vector is called for short pC) is contrast, and 1,2,3 is that 3 paddy rice cross expression strain.2 weeks later leaf morphologies of inoculation bacterial leaf spot, illustrate that wheat TaCPK2 gene participates in paddy disease-resistant reaction.
Embodiment 7: wheat calcium-dependent protein kinase gene TaCPK2 increases resistance---the strain phase of paddy rice to bacterial leaf spot
The rice conversion plant obtaining from embodiment 5 is cultivated together with the while large Tanaka with its empty carrier (wild-type is that sky educates 131), until the strain phase.Fig. 5 shows wheat calcium-dependent protein kinase gene TaCPK2 rice transformation, causes strain phase paddy rice to increase the resistance of bacterial leaf spot.As shown in Figure 5, empty carrier (empty vector is called for short pC) is contrast, and 1,2,3 is that 3 paddy rice cross expression strain.2 weeks later leaf morphologies of inoculation bacterial leaf spot, illustrate that wheat TaCPK2 gene participates in paddy disease-resistant reaction.
Embodiment 8: statistical result showed: wheat calcium-dependent protein kinase gene TaCPK2 increases the resistance of rice seedling to bacterial leaf spot
The withered spot length of blade from embodiment 6 obtains rice conversion plant inoculation bacterial leaf spot and the ratio of blade total length are added up, and result shows that wheat calcium-dependent protein kinase gene TaCPK2 increases the resistance of paddy rice to bacterial leaf spot really.As shown in Figure 6, empty carrier (pC) is contrast, and 1,2,3 is the statistical value of 3 strains (9 blades of each strain).Bacterial leaf spot infects ratio and is respectively: pC, 52%; 1,24%; 2,29%; 3,11%.Data presentation, transfer-gen plant obviously reduces than contrast scab ratio, and bacterial leaf spot resistance is increased.
Embodiment 9: measure the expression amount that TaCPK2 crosses the middle TaCPK2 of expression plant (paddy rice) and OsCPK13
Utilize RT-PCR to measure the expression amount that TaCPK2 crosses the middle TaCPK2 of expression plant (paddy rice) and OsCPK13, result as shown in Figure 7.OsCPK13 gene in paddy rice and the similarity of TaCPK2 gene order are the highest.From the expression paddy rice that crosses of embodiment 6 and embodiment 7 acquisitions, carry out RT-PCR evaluation, at paddy rice Tubulin, detect under the quality of cDNA and the basically identical condition of concentration, TaCPK2 crosses the expression that TaCPK2 detected in expression plant (paddy rice), and in empty carrier (pC), nothing is expressed; And the native gene OsCPK13 expression amount in paddy rice is substantially constant, crossing of TaCPK2 gene expressed the expression that does not affect paddy rice native gene OsCPK13.So the resistant effect that embodiment 6 and embodiment 7 express plant is excessively to produce owing to crossing the wheat TaCPK2 gene of expressing.
Embodiment 10: wheat calcium-dependent protein kinase gene TaCPK2 paddy rice crosses expresses plant and the output of paddy rice not have to affect substantially not connecing under bacterium condition.
The paddy rice species test result obtaining from embodiment 7 is as shown in Figure 8: three transgenic lines (1,2,3) are compared with empty carrier (pC), and tiller number (A) and grain number per spike (B) reduce slightly; Heavy (D) is substantially constant for plant height (C) and simple grain, illustrate that wheat calcium-dependent protein kinase gene TaCPK2 paddy rice crosses to express plant and the output of paddy rice is not had to affect substantially not inoculating under bacterial leaf spot condition.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvement to it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (3)
1. the application of wheat TaCPK2 albumen in Resistant breeding process, is characterized in that, described disease-resistant finger resisting bacterial leaf-blight.
2. application as claimed in claim 1, is characterized in that, the aminoacid sequence of described wheat TaCPK2 albumen is as shown in SEQ ID No.2.
3. the application as described in claim 1-2 any one, is characterized in that, described plant is wheat or paddy rice.
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| CN105132517B (en) * | 2015-08-28 | 2019-08-23 | 河南农业大学 | Method of the joint identification wheat to Bipolaris Sorokiniana Dark kernel and leaf blight resistance |
| CN110627885B (en) * | 2018-05-30 | 2021-01-15 | 中国农业科学院作物科学研究所 | Wheat powdery mildew resistance gene and application thereof |
| CN111197035B (en) * | 2020-01-08 | 2022-09-13 | 西北农林科技大学 | Powdery mildew resistant grape calcium-dependent protein kinase gene VpCDPK13 and its application |
| CN111118042B (en) * | 2020-01-08 | 2022-08-30 | 西北农林科技大学 | Powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK9 and application thereof |
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