CN103305486A - Application of wheat TaCPK2 protein in plant disease-resistant breeding - Google Patents

Abstract

The invention discloses the application of wheat TaCPK2 protein in the process of plant disease resistance breeding. The advantage of the present invention is that the wheat TaCPK2 gene of the present invention belongs to the genes of the CDPK gene family involved in plant defense against pathogens, and inhibiting the expression of the TaCPK2 gene in wheat can significantly affect the resistance of wheat to powdery mildew, while overexpression in rice TaCPK2 gene can significantly increase the resistance of transgenic rice to bacterial blight. It can be used to solve the problems of plant yield reduction caused by fungal or bacterial diseases such as powdery mildew and bacterial blight.

Description

Application of Wheat TaCPK2 Protein in Plant Disease Resistance Breeding

technical field

The invention relates to the field of genetic engineering, in particular to the application of a calcium-dependent protein kinase gene in disease resistance engineering.

Background technique

In recent years, due to the extensive use of fungicides, although there have been no large-scale outbreaks of crop diseases, excessive reliance on pesticides has not only increased production costs, but also caused large-scale environmental pollution, seriously threatening agricultural ecological security. The most economical, safe and effective way to control the prevalence of crop diseases is to clone crop disease-resistant genes and apply them to crop disease-resistant genetic breeding by means of biotechnology. However, due to the interaction between disease-resistant genes and avirulent genes, the specific resistance of varieties to races is expressed, and plant disease resistance is often overcome by changes in the physiological races of pathogen populations. Some disease-resistant genes have not yet been cloned. , its disease resistance is lost due to changes in the toxicity of the bacterial population. It can be seen that the use of disease-resistant genes to breed disease-resistant varieties is also constantly facing new challenges. People have instead begun to screen for genes important for broad-spectrum, long-lasting disease resistance and to breed crop varieties with long-lasting disease resistance.

Calcium-dependent protein kinase (CDPK) is a calcium ion (Ca ²⁺ ) sensor unique to plants and protozoa. Abiotic stress, pathogens, hormones, and light stimuli can all lead to changes in plant endogenous Ca ²⁺ concentrations (Knight and Knight, 2001; Sanders, 2002), and CDPK can recognize changes in Ca ²⁺ concentrations and change downstream proteins (such as transcription factors ) phosphorylation status, affecting gene expression patterns (Sanders et al., 1999). The CDPK protein contains four structural domains, namely the N-terminal variable region (N), the protein kinase domain (kinase, K), the autoinhibitory region (autoinhibitory region, A) and the calmodulin-like domain (CaM-like) (Cheng et al., 2002; Hrabak et al., 2003). The CaM-like domain contains the EF hands functional domain that can bind Ca ²⁺ . When Ca ²⁺ binds to the CaM-like domain of CDPK, the inhibition of the kinase domain by the autoinhibition region is released, and CDPK is activated. CDPKs play the role of key regulators in multiple signal transmission pathways in plants.

As an early signal factor, CDPK is located upstream of the signal transmission pathway and plays an important role in plant pathogen defense (Cheng et al., 2002). Tobacco NtCPK2 was found to be a component of the R gene-mediated signaling pathway (Romeis et al., 2001). In barley, transient expression of HvCDPK4 induces the spread of tobacco mesophyll cell death similar to allergic reactions, which is not conducive to fungal invasion; while transient expression of HvCDPK3 is conducive to fungal invasion (Freymark et al., 2007). The HvCDPK3 and HvCDPK4 genes in barley play an antagonistic role in the process of controlling the entry of host cells in the early stages of powdery mildew. OsCPK13 (OsCDPK7) in rice plays an important role in adversity environments such as salt, drought and low temperature (Saijo et al., 2000; Komatsu et al., 2007), and it was overexpressed in sorghum, but it showed disease resistance Related traits (Mall et al., 2011). StCDPK5 induces the phosphorylation of StRBOHB and regulates oxidative burst in potato (Kobayashi et al., 2007), which may be involved in the plant defense response to pathogens.

Although the wheat CDPK family has been reported (Li et al., 2008), the biological function of TaCPK2 has not been clearly elucidated.

Contents of the invention

In order to solve the above problems, the object of the present invention is to provide an application of wheat TaCPK2 protein in plant disease resistance breeding.

In order to achieve the above object, the present invention provides a wheat TaCPK2 protein whose amino acid sequence is as shown in SEQ ID No.2.

The invention provides the application of wheat TaCPK2 protein in the process of plant disease resistance breeding.

The disease is preferably powdery mildew or bacterial blight; the plant is preferably wheat or rice.

The application of the wheat TaCPK2 protein provided by the invention in regulating plant disease resistance. The application refers to overexpressing the wheat TaCPK2 gene, regulating the expression changes of genes related to the SA and JA pathways, so as to make the plants produce disease resistance.

The above-mentioned technical scheme has the following advantages:

The wheat TaCPK2 gene of the present invention belongs to the gene of the CDPK gene family involved in plant defense against pathogens, inhibiting the expression of the TaCPK2 gene in wheat can significantly affect the resistance of wheat to powdery mildew, and overexpressing the TaCPK2 gene in rice can significantly increase the transgenic Rice is resistant to bacterial blight. It can be used to solve the problems of plant yield reduction caused by fungal or bacterial diseases such as powdery mildew and bacterial blight.

Description of drawings

Fig. 1 is the cloning intermediate vector pEASY-T1 Simple of embodiment 1 of the present invention and embodiment 3 wheat TaCPK2 gene;

Fig. 2 is the phenotype observation that the disease resistance of the wheat calcium-dependent protein kinase TaCPK2 gene VIGS of embodiment 4 of the present invention reduces;

Fig. 3 is the plant expression vector pCUbi1390 of Example 5 of the present invention;

Fig. 4 is that the wheat calcium-dependent protein kinase gene TaCPK2 of Example 6 of the present invention increases the resistance of rice (seedling stage) to bacterial blight;

Fig. 5 is that the wheat calcium-dependent protein kinase gene TaCPK2 of Example 7 of the present invention increases the resistance of rice (adult plant stage) to bacterial blight;

Fig. 6 is the quantitative statistical result of Example 8 of the present invention showing that the wheat calcium-dependent protein kinase gene TaCPK2 increases the resistance of rice at the seedling stage to bacterial blight;

Fig. 7 is the RT-PCR positive identification of overexpressed rice plants in Example 9 of the present invention and the expression of endogenous genes remains unchanged;

Fig. 8 is the test result of wheat calcium-dependent protein kinase TaCPK2 gene overexpressed in rice without inoculation in Example 10 of the present invention.

Among them, the empty vector (pC for short) is the control, and 1, 2, and 3 are the three rice overexpression lines.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modifications or replacements made to the methods, steps or conditions of the present invention belong to the protection scope of the present invention.

The biological materials involved in the present invention are Chinese spring wheat, durum wheat DR147, wheat Am6, and Beijing 837 are all known public varieties. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Example 1 Cloning of full-length gene of wheat calcium-dependent protein kinase TaCPK2

ATGGGCAACGCATGCGGCGGT(如SEQ ID No.4所示)和反向引物GCATCTAGATTGCACCAGGTGCGTC(如SEQ ID No.5所示)从中国春小麦(Triticum aestivum L.)叶的cDNA中克隆TaCPK2基因的编码区序列；Use primers with restriction enzyme sites, 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 wheat (Triticum aestivum L.) leaf;

PCR program: 94°C, 5 minutes; 94°C, 30 seconds; 55°C, 30 seconds; 72°C, 90 seconds; repeat 35 times; 72°C, 10 minutes.

PCR system: 2×EasyTaq PCR SuperMix (Quanshijin Company) 25μl;

Forward primer (10μM) 2μl;

Reverse primer (10μM) 2μl;

DNA template 5 μl;

Make up 50 μl of double distilled water.

After the PCR product was gel-cut, recovered and purified, it was cloned and connected to the pEASY-T1Simple vector according to the pEASY-T1 Simple Cloning Kit cloning method provided by Beijing Quanshijin Biotechnology Co., Ltd. (Figure 1), and the ligated product was transformed into E. coli DH5α, and amplified in it The positive clones were sequenced and screened to obtain TaCPK2, the cDNA sequence of which is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2.

Embodiment 2: Structural analysis of protein encoded by wheat calcium-dependent protein kinase gene TaCPK2

The structural analysis of the protein encoded by the wheat calcium-dependent protein kinase gene TaCPK2 showed that the protein encoded by this gene has the same conserved enzyme active site and structural domain as the CDPK protein domain in other plants. Phylogenetic analysis showed that the protein encoded by wheat TaCPK2 gene had the highest similarity with HvCDPK4 protein in barley, which was 95.9%. HvCDPK4 induces the spread of mesophyll cell death, which is not conducive to the invasion of fungi, and plays a role in resisting the invasion of powdery mildew. Therefore, the wheat TaCPK2 gene and the HvCDPK4 gene in barley have similar functions in resisting the invasion of powdery mildew.

Embodiment 3: VIGS vector and VIGS plant of wheat calcium-dependent protein kinase gene TaCPK2

TGTCCTTTGATGGGCAACGCA(如SEQ ID No.6所示)和反向引物CCGCGTGGCCGTCCGTCTT(如SEQ ID No.7所示)从中国春小麦(Triticum aestivum L.)叶的cDNA中进行PCR扩增，PCR程序：94℃，5分钟；94℃，30秒；56℃，30秒；72℃，30秒；重复35次；72℃，10分钟。Use primers with restriction enzyme sites, forward primers

TGTCCTTTGATGGGCAACGCA (as shown in SEQ ID No.6) and reverse primer CCGCGTGGCCGTCCGTCTT (as shown in SEQ ID No.7) was amplified by PCR from the cDNA of Chinese spring wheat (Triticum aestivum L.) leaves, PCR program: 94°C, 5 minutes; 94°C, 30 seconds; 56°C, 30 seconds; 72°C, 30 seconds; repeat 35 times; 72°C, 10 minutes.

PCR system: 2×EasyTaq PCR SuperMix (Quanshijin Company) 25μl;

Forward primer (10μM) 2μl;

Reverse primer (10μM) 2μl;

DNA template 5 μl;

Make up 50 μl of double distilled water.

After the PCR product was gel-cut, recovered and purified, it was cloned and connected to the pEASY-T1 Simple vector according to the pEASY-T1 Simple Cloning Kit cloning method provided by Beijing Quanshijin Biotechnology Co., Ltd. (Figure 1), and the ligated product was transformed into Escherichia coli DH5α. Amplified, positive clones were sequenced and screened to obtain the cDNA fragment of TaCPK2, the cDNA sequence of which is shown in SEQ ID NO.3. The barley stripe mosaic virus (BSMV) vector consists of three components: BSMV-α, BSMV-β, and BSMV-γ (Holzberg et al., 2002; Zhao Dan et al., 2011). Digest the correctly sequenced pEASY-T1Simple vector and BSMV-γ vector with NheI of NEB, recover the digested TaCPK2 (190bp) gene fragment and BSMV-γ vector fragment, and connect them with T4 DNA ligase (NEB). The gene fragment was ligated in reverse to the multiple cloning site of the BSMV-γ vector. The ligation product was transformed into Escherichia coli DH5α competent, and the positive clones were screened by bacterial liquid PCR and sequenced to verify that the vector plasmid with the correct sequence was obtained as the recombinant vector BSMV-γ:TaCPK2.

Green fluorescent protein (Green Fluorescent Protein, GFP) is derived from marine organisms jellyfish, its gene can be expressed in heterologous tissues and produce fluorescence, GFP cDNA open reading frame length is about 714bp, encoding 238 amino acid residues, the first peptide chain internal Serine-dehydrotyrosine-glycine at position 65-67 forms a chromogenic gene through self-cyclization and oxidation, which emits green fluorescence under long ultraviolet wavelength or blue light irradiation. Green fluorescent protein is one of the commonly used reporter genes. The reason why green fluorescent protein was used as a control in this study is that the gene does not have any homologous genes in plants, so the BSMV vector carrying GFP will not silence any genes in plants, and can be used as a control for gene down-regulation.

In VIGS experiments, BSMV:GFP is often used as a negative control (Li et al., 2011; Cao et al., 2011). The vector construction process is the same as that of the BSMV-γ:TaCPK2 vector, that is, the cloned GFP gene fragment and the BSMV-γ vector fragment are connected with T4DNA ligase (NEB), and transformed into E. coli competent cells, and sequenced and screened to obtain the correct The vector plasmid is the BSMV-γ:GFP recombinant vector.

Method steps for obtaining VIGS plants:

1) Extract the plasmids of BSMV-α, BSMV-β, BSMV-γ:TaCPK2 and BSMV-γ:GFP, BSMV-α, BSMV-γ:TaCPK2 and BSMV-γ:GFP are digested with Mlu1, and BSMV-β is digested with Spe1 Enzyme digestion, 37°C, 8h. For linearization treatment, the 50μl NEB enzyme digestion system is as follows:

Mlu1:

10×NEB buffer: 5 μl

Plasmid: ≤2μg

Mlu1: 1 μl

Sterilized double distilled water: to 50μl

Spe1:

10×NEB buffer: 5 μl

Plasmid: ≤2μg

100×BSA: 0.5 μl

Mlu1: 1 μl

Sterilized double distilled water: to 50μl

2) Ethanol precipitation

Add 350 μl DEPC (Diethyl pyrocarbonate) H ₂ O to 150 μl linearized plasmid, add 500 μl phenol chloroform (volume ratio of phenol and chloroform is 1:1), and centrifuge at 12000 rpm for 30 min. Take 450 μl of supernatant, add 45 μl of NaAc (PH 5.2, 3M), add 1 ml of cold (4°C) absolute ethanol, and mix well. -20°C overnight. Centrifuge at 12000rpm for 30min, discard the supernatant, wash twice with 1ml 70% ethanol, dry in the air, add 30μl DEPC H ₂ O to dissolve. The concentration should be ≥400ng/μl.

3) The in vitro transcription kit uses AmpliCap-Max ^TM T7 and T3 High Yield Message Maker Kits (Epicentre, USA) provided by Beijing GBI Company

10μl reaction system:

Template: 3 μl

10×transcription buffer: 1μl

Cap/NTP master mix: 4μl

100mM dithiothreitol: 1 μl

T7 enzyme: 1 μl

42°C, 3h.

4) Virus inoculation

Take 10 μl of BSMV-α, BSMV-β, BSMV-γ:TaCPK2 (or BSMV-γ:GFP) transcripts and mix them to make a total of 30 μl, add 3 times the volume (90 μl) of DEPC H ₂ O, the total volume reaches 120 μl, and then Add double volume (120 μl) of GKP buffer to prepare 240 μl TaCPK2 and GFP inoculated virus mixture.

_The wheat material used for virus infection was the powdery mildew resistant near-isogenic line PmAm6/ _Beijing 837*BC ₅ F ₃ The hexaploid wheat Am6 was artificially synthesized with diploid Aegilopstaushii Ae39 as the female parent and the male parent. With Am6 as the donor parent and Beijing 837 as the recurrent parent, the powdery mildew resistance single was screened from the hybrid F1 strains, backcross with Beijing 837, and select disease-resistant single plants from the progeny of each backcross. Select homozygous disease-resistant single plants from the F ₃ family of the 5th generation of backcross, that is, PmAm6/Beijing837*BC ₅ F ₃ ). When the second leaf of the wheat seedlings was flattened (14d), the inoculation began. Wearing rubber gloves that have just been opened, take 5-10 μl of the inoculation solution on the belly of the index finger with a treated RNase-free pipette tip, fix the base of the wheat seedling with one hand, and gently rub the second wheat leaf back and forth with the thumb and index finger with the other hand .

After inoculation, spray DEPC H ₂ O to the wheat seedlings, cover them with plastic wrap, keep them moist for 24 hours (high humidity is conducive to virus invasion), and then uncover them. The temperature can be between 20-32°C (rising temperature is not conducive to virus invasion, and the leaves grow quickly).

After 6-8 days, virus spots were produced on the third leaf, which means that the virus inoculation was successful, and VIGS plants of BSMV:TaCPK2 and BSMV:GFP were obtained.

Example 4: VIGS experiment proves that TaCPK2 gene is involved in powdery mildew resistance response

The BSMV obtained in Example 3: TaCPK2 and BSMV: GFP plants are cultivated together in an incubator until the fourth leaf is flattened, and the leaves of the plants with the virus morbidity phenotype are cut off, placed on the benzimidazole medium and inoculated with powdery mildew E09 and cultivated for one week It can be clearly seen that the down-regulation of TaCPK2 gene expression in BSMV:TaCPK2 plants leads to powdery mildew infection, and there are white spore piles visible to the naked eye on the leaf surface, while the control BSMV:GFP plants are still resistant to powdery mildew because the TaCPK2 gene expression has not changed. The leaf surface is clean. It was concluded that TaCPK2 gene was involved in powdery mildew resistance. As shown in Figure 2, Bj grows spore piles after being inoculated with powdery mildew for susceptible materials, and is used as a susceptible control; MOCK is inoculated with GKP buffer for resistant powdery mildew materials, and does not infect powdery mildew; BSMV:GFP is for inoculated virus BSMV:GFP plants, No powdery mildew infection; BSMV:TaCPK2 was inoculated with virus BSMV:TaCPK2 plants, locally infected with powdery mildew, proving that TaCPK2 gene is involved in powdery mildew resistance.

Embodiment 5: Wheat calcium-dependent protein kinase gene TaCPK2 plant expression vector

The correct TaCPK2 sequence obtained from Example 1, using two restriction sites KpnI and SpeI, obtains the full-length sequence TaCPK2 constructed on the pEASY-T1 Simple (Beijing Quanshijin Biotechnology Co., Ltd.) carrier, and expresses it in plants Carrier pCUbi1390 (Penget al., 2009) (Fig. 3) after carrying out the same digestion, according to the use method of T4 DNA ligase of NEB company, the two fragments obtained by restriction enzyme digestion are ligated, and according to the method among the embodiment 3 The ligation product was transformed into Escherichia coli DH5α and multiplied in it, and the positive clone was sequenced to obtain the overexpression vector of TaCPK2. The obtained TaCPK2 overexpression vector is transformed into rice by an Agrobacterium-mediated method to obtain transformed plants. The selection marker in plants is hygromycin.

Application example 6: Wheat calcium-dependent protein kinase gene TaCPK2 increases rice resistance to bacterial blight --- seedling stage

The transformed rice plants obtained in Example 5 and their empty vector (wild-type Kongyu 131) were cultivated together in the field until the adult plant stage. Figure 4 shows that transformation of rice with wheat calcium-dependent protein kinase gene TaCPK2 leads to increased resistance of rice seedlings to bacterial blight. As shown in Figure 4, the empty vector (pC for short) is the control, and 1, 2, and 3 are the three rice overexpression lines. Leaf phenotypes 2 weeks after inoculation with bacterial blight indicated that the wheat TaCPK2 gene was involved in rice disease resistance.

Example 7: Wheat calcium-dependent protein kinase gene TaCPK2 increases rice resistance to bacterial blight --- adult plant stage

The rice transformed plants obtained from Example 5 and their empty vectors (Kongyu 131 as the wild type) were cultivated together in the field until the adult plant stage. Figure 5 shows that transformation of rice with wheat calcium-dependent protein kinase gene TaCPK2 leads to increased resistance of rice to bacterial blight at the adult plant stage. As shown in Figure 5, the empty vector (pC for short) is the control, and 1, 2, and 3 are the three rice overexpression lines. Leaf phenotypes 2 weeks after inoculation with bacterial blight indicated that the wheat TaCPK2 gene was involved in rice disease resistance.

Example 8: Statistical results show that the wheat calcium-dependent protein kinase gene TaCPK2 increases the resistance of rice seedlings to bacterial blight

The ratio of the length of the dead spots on the leaves of the transformed rice plants inoculated with bacterial blight to the total length of the leaves was obtained from Example 6, and the results showed that the wheat calcium-dependent protein kinase gene TaCPK2 did increase the resistance of rice to bacterial blight. As shown in Figure 6, the empty vector (pC) is the control, and 1, 2, and 3 are the statistical values of 3 lines (9 leaves for each line). The proportions of bacterial blight infection were: pC, 52%; 1, 24%; 2, 29%; 3, 11%. The data showed that the lesion ratio of the transgenic plants was significantly smaller than that of the control, and the resistance to bacterial blight was increased.

Example 9: Determination of the expression levels of TaCPK2 and OsCPK13 in TaCPK2 overexpression plants (rice)

The expression levels of TaCPK2 and OsCPK13 in TaCPK2 overexpressed plants (rice) were measured by RT-PCR, and the results are shown in FIG. 7 . The sequence similarity between OsCPK13 gene and TaCPK2 gene in rice is the highest. The overexpressed rice obtained from Example 6 and Example 7 was identified by RT-PCR. Under the condition that the quality and concentration of the cDNA detected by rice Tubulin were basically the same, the expression of TaCPK2 was detected in the overexpressed plant (rice) of TaCPK2. Empty vector There was no expression in (pC); and the expression level of the endogenous gene OsCPK13 in rice was basically unchanged, that is, the overexpression of the TaCPK2 gene did not affect the expression of the rice endogenous gene OsCPK13. Therefore, the disease resistance of the overexpressed plants in Examples 6 and 7 is due to the overexpressed wheat TaCPK2 gene.

Example 10: Plants overexpressing wheat calcium-dependent protein kinase gene TaCPK2 in rice have basically no effect on rice yield under the condition of no inoculation.

The rice test result that obtains from embodiment 7 is as shown in Figure 8: three transgenic lines (1,2,3) are compared with empty vector (pC), tiller number (A) and panicle number (B) slightly decreased; plant height (C) and single-grain weight (D) were basically unchanged, indicating that rice overexpression plants of wheat calcium-dependent protein kinase gene TaCPK2 basically had no effect on rice yield under the condition of not being inoculated with bacterial blight.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (4)

1. the application of wheat TaCPK2 albumen in the Resistant breeding process.

2. application as claimed in claim 1 is characterized in that, described disease is Powdery Mildew or bacterial leaf-blight.

3. application as claimed in claim 1 is characterized in that, the aminoacid sequence of described wheat TaCPK2 albumen is shown in SEQ ID No.2.

4. as any described application of claim 1-3, it is characterized in that described plant is wheat or paddy rice.

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