CN114835789B - Wheat powdery mildew resistance related protein TaGLP-7A, and coding gene and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a wheat powdery mildew resistance related protein TaGLP-7A, and a coding gene and application thereof. The invention relates to a new clone obtained from wheat variety Bainong 207GLPGenes, which are designated asTaGLP‑7A. The invention is constructed to silence wheatTaGLP‑7ABarley streak mosaic virus inducible vector of gene, decreaseTaGLP‑7AExpression of the Gene is silencedTaGLP‑7AWheat plants, silencingTaGLP‑7AWheat inoculation powdery mildewAfter that, powdery mildew resistance is significantly reduced. Furthermore, the present invention further constructs overexpressionTaGLP‑7AWheat stable genetic transformation plant and overexpressionTaGLP‑7AThe powdery mildew resistance level of the wheat plants is obviously improved, thereby indicatingTaGLP‑7AAfter the expression quantity in the infected wheat plant is improved, the powdery mildew resistance of the infected wheat plant can be obviously improved, so that the gene can be used for cultivating powdery mildew resistant wheat by utilizing a genetic engineering means.

Description

Wheat powdery mildew resistance-related protein TaGLP-7A and its encoding genes and applications

Technical field

The invention relates to the field of biotechnology, specifically to wheat powdery mildew resistance-related protein TaGLP-7A and its encoding gene and application.

Background technique

Wheat (Triticum aestivum L.) is the main source of human food calories. China is the largest country in wheat cultivation and production, and it plays an important role in ensuring domestic food security. Wheat powdery mildew is a worldwide disease caused by the biotrophic obligate parasitic fungus Blumeria graminis f.sp. Tritici. It usually causes 13%-34% yield loss and is serious during the heading and grain filling stages. It can lead to 50% yield loss, and in some extreme cases, it can cause the leaves to dry out and even the plant to die. With the variation in the virulence structure of the virus, climate change and the implementation of certain farming methods, the incidence rate of wheat powdery mildew in winter wheat areas in China is increasing year by year, and there is a trend of expanding eastward and moving northward.

Breeding resistant varieties is one of the most cost-effective and environmentally friendly ways to reduce wheat powdery mildew losses. Since powdery mildew resistance breeding in China in the last century, only a few disease-resistant genes such as Pm2, Pm3, Pm4, Pm6, Pm8, and Pm21 have been introduced in China. China's wheat production has been widely used, and the resistance of most race-specific disease resistance genes has been gradually overcome by new toxic races, gradually losing its application value in production, resulting in widespread use in actual production. There are not many broad-spectrum powdery mildew resistance genes. Therefore, it is urgent to discover new long-lasting broad-spectrum high resistance genes and explore new ways to breed wheat for powdery mildew resistance to improve wheat's long-lasting broad-spectrum resistance to powdery mildew.

Germin-like proteins (GLPs) are members of the Cupin superfamily and are often composed of two exons and one intron. The encoded protein contains approximately 220 amino acids and contains a conserved Cupin at the C-terminus. domain. GLPs play an important role in plant defense responses. Some GLPs have oxalate oxidase (OXO) or superoxide dismutase (SOD) activity. They catalyze the H ₂ O ₂ produced by oxalic acid, etc. and promote the cross-talk of cell walls at the site of pathogenic bacteria infection. As an important intracellular signal, H ₂ O ₂ can also activate a series of plant defense-related signal transduction pathways to prevent the invasion of pathogenic bacteria. Research shows that peanut AhGLP shows a unique response pattern to stresses such as Aspergillus flavus, mosaic disease and rust; GhABP19 in upland cotton plays an important role in regulating cotton's resistance to Verticillium wilt; overexpression of OsGLP2- 1 rice has significantly enhanced resistance to rice blast and bacterial blight. The above studies show that GLP genes play an important role in plant disease resistance. However, there are currently few reports on the cloning and functional studies of this type of genes in wheat powdery mildew research.

Contents of the invention

In view of the problems and deficiencies existing in the prior art, the purpose of the present invention is to provide wheat powdery mildew resistance-related protein TaGLP-7A and its encoding gene and application.

In a first aspect, the invention provides a protein.

The protein provided by the invention is derived from Triticum aestivum L., and is named TaGLP-7A, which is the following protein a) or b) or c) or d):

a) The amino acid sequence is the protein shown in SEQ ID NO.2;

b) A fusion protein obtained by connecting a tag to the N-terminal and/or C-segment of the protein shown in SEQ ID NO.2;

c) A protein with the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO.2;

d) A protein that has 85% or more homology with the amino acid sequence shown in SEQ ID NO. 2 and has the same function.

Among them, SEQ ID NO. 2 consists of 212 amino acid residues.

In order to facilitate the purification of the protein in a), the tag shown in Table 1 can be connected to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO. 2 in the sequence listing.

Table 1 Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

For the protein in c) above, the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution, deletion and/or addition of no more than 10 amino acid residues.

The protein in c) above can be synthesized artificially, or its encoding gene can be synthesized first and then biologically expressed.

The gene encoding the protein in c) above can be obtained by deleting one or a few nucleotide codons in the DNA sequence shown in SEQ ID NO. 1, and/or performing a missense mutation of one or several base pairs. , and/or the coding sequence of the tag shown in Table 1 is connected to its 5′ end and/or 3′ end.

In a second aspect, the present invention provides a nucleic acid molecule encoding TaGLP-7A protein.

The coding sequence of the nucleic acid molecule is shown in SEQ ID NO.1.

In a third aspect, the present invention provides recombinant vectors, expression cassettes, transgenic cell lines, recombinant bacteria or recombinant viruses containing the nucleic acid molecules described in the second aspect.

In a third aspect, the present invention provides new uses for the protein described in the first aspect, the nucleic acid molecule described in the second aspect, or the recombinant vector or expression cassette, transgenic cell line, recombinant bacteria or recombinant virus described in the third aspect.

The present invention provides the use of the TaGLP-7A protein or nucleic acid molecule or recombinant vector or expression cassette or transgenic cell line or recombinant bacteria or recombinant virus described in the first aspect in regulating plant powdery mildew resistance.

The present invention also provides the use of the TaGLP-7A protein or nucleic acid molecule or recombinant vector or expression cassette or transgenic cell line or recombinant bacteria or recombinant virus described in the first aspect in cultivating transgenic plants with reduced powdery mildew resistance.

The present invention also provides the use of the TaGLP-7A protein or nucleic acid molecule or recombinant vector or expression cassette or transgenic cell line or recombinant bacteria or recombinant virus described in the first aspect in transgenic plants with improved powdery mildew resistance.

In the above application, the plant is a monocotyledonous plant or a dicotyledonous plant. The dicotyledonous plant may be Arabidopsis thaliana, and the monocotyledonous plant may be wheat (Triticum aestivum L.), corn, etc.

In a fourth aspect, the present invention provides a method for cultivating transgenic plants with improved powdery mildew resistance.

The method for cultivating transgenic plants with improved powdery mildew resistance provided by the invention includes the steps of increasing the expression amount and/or activity of TaGLP-7A protein in recipient plants to obtain transgenic plants; the powdery mildew resistance of the transgenic plants is higher than The recipient plant.

In the above method, the method of increasing the expression level and/or activity of TaGLP-7A protein in the recipient plant is: overexpressing the TaGLP-7A protein in the recipient plant.

In the above method, the overexpression method is to introduce the gene encoding the TaGLP-7A protein into the recipient plant. Preferably, the nucleotide sequence of the gene encoding TaGLP-7A protein is the DNA molecule shown in SEQ ID NO.1.

According to the above method, the recipient plant is a monocotyledonous plant or a dicotyledonous plant. The dicotyledonous plant may specifically be Arabidopsis thaliana, and the monocotyledonous plant may specifically be wheat (Triticumaestivum L.), corn, etc.

In a fifth aspect, the present invention provides a method for cultivating transgenic plants with reduced powdery mildew resistance.

The method for cultivating transgenic plants with reduced powdery mildew resistance provided by the invention includes the steps of inhibiting the expression and/or activity of TaGLP-7A protein in recipient plants to obtain transgenic plants; the powdery mildew resistance of the transgenic plants is lower than The recipient plant.

In the above method, the method of inhibiting the expression amount and/or activity of TaGLP-7A protein in the recipient plant is: introducing a substance that inhibits the expression of TaGLP-7A protein into the recipient plant to obtain a transgenic plant, and the transgenic plant has Powdery mildew resistance is lower than in the recipient plants.

According to the above method, the recipient plant is a monocotyledonous plant or a dicotyledonous plant. The dicotyledonous plant may specifically be Arabidopsis thaliana, and the monocotyledonous plant may specifically be wheat (Triticumaestivum L.), corn, etc.

The positive beneficial effects achieved by the present invention are:

The present invention clones a germin-like gene with a Cupin domain from the wheat variety Bainong 207, names it TaGLP-7A, and constructs a virus-inducible vector for silencing the TaGLP-7A gene in wheat, reducing TaGLP- Expression of the 7A gene resulted in silenced TaGLP-7A wheat. After the silenced TaGLP-7A wheat was inoculated with powdery mildew bacteria, the wheat plants showed an obvious phenotype of reduced powdery mildew resistance, indicating that the TaGLP-7A gene in wheat is suppressed in plants through silencing means. The expression can reduce the resistance of wheat to powdery mildew. Moreover, the present invention further obtains a transgenic line that stably overexpresses TaGLP-7A by bombarding common wheat Bainong 207 immature embryos with a gene gun method, and conducts powdery mildew resistance identification on the T ₁ generation plants of the transgenic line that overexpresses TaGLP-7A. It was found that overexpression of TaGLP-7A can significantly improve the resistance of transgenic lines to wheat powdery mildew. The above research results show that the TaGLP-7A gene positively regulates wheat powdery mildew resistance, and overexpression of the TaGLP-7A gene in wheat can improve wheat resistance to powdery mildew.

Description of the drawings

Figure 1 shows TaGLP-7A gene expression analysis at different time points after Bainong 207 was inoculated with wheat powdery mildew; the abscissa represents the different time points after infection of Bainong 207 with a wheat powdery mildew mixed strain (h: hours postinoculation); the ordinate represents Represents the relative expression of TaGLP-7A gene in inoculated leaves;

Figure 2 shows the results of TaGLP-7A gene expression detection in silenced TaGLP-7A wheat; CK indicates infection with BSMV: γ empty control wheat plants; 1 and 2 both indicate infection with BSMV: γ-TaGLP-7A wheat plant;

Figure 3 shows the phenotypes produced by VIGS silencing the wheat Bainong 207 gene. Among them, BSMV:γ and BSMV:TaGLP-7A respectively represent the phenotypes of infected BSMV:γ empty control and BSMV:γ-TaGLP-7A plant leaves inoculated with powdery mildew;

Figure 4 is a schematic diagram of the map structure of TaGLP-7A overexpression vector PBI220:TaGLP-7A;

Figure 5 shows the PCR identification of T ₀ generation of PBI220:TaGLP-7A transgenic plants; lane 1 is Marker: DL2000 DNA standard molecular weight; lane 2 plasmid: PBI220:TaGLP-7A positive control; lane 3 acceptor Bainong 207 negative control; lane 4 Water control; lane 5TaGLP-7A-T ₀ -42 negative control; lanes 6, 7, and 8 are positive for TaGLP-7A-T ₀ -OE1, TaGLP-7A-T ₀ -OE2, and TaGLP-7A-T ₀ -OE3 respectively. Amplification results of transgenic plants;

Figure 6 shows the expression analysis of TaGLP-7A gene in the leaves of TaGLP-7A-T ₁ -OE1, TaGLP-7A-T ₁ -OE2, and TaGLP-7A-T ₁ -OE3 transgenic positive lines; the values of qRT-PCR It is the expression level of relative receptor material WT (wheat) with TaTubulin as the internal reference, CK represents the transgenic negative control TaGLP-T ₁ -42; ** represents the significant difference analysis by one-way ANOVA LSD method (P<0.01) ;

Figure 7 shows the powdery mildew resistance identification results of the T ₁ generation positive plants transformed with Bainong 207 with TaGLP-7A; among them, T ₁ -TaGLP-OE1 represents the resistance identification of the TaGLP-7A positive strain T ₁ -OE1; T ₁ -TaGLP-7A-OE2 represents the resistance identification of TaGLP-7A positive strain T ₁ -OE2 strain; T1-TaGLP-7A-OE3 represents the resistance identification of TaGLP-7A positive strain T ₁ -OE3 strain; Negative represents Resistance identification of transgenic negative control lines.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test reagents used in the following examples were all purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following examples were repeated three times, and the results were averaged.

Example 1: Cloning of TaGLP-7A gene

1. Obtaining cDNA

Bainong 207 (Bainong 207 is a hybrid of Zhoumai 16/Bainong 64 selected by Henan University of Science and Technology through traditional breeding, integrating multiple excellent traits such as high yield, high quality, disease resistance and wide adaptability) An excellent variety, Bainong 207, is highly susceptible to powdery mildew in the seedling stage and moderately resistant in the adult stage. Leaves induced by powdery mildew for 24 hours were used as raw materials. The total wheat leaf total was extracted according to the instructions of the plant RNA extraction kit (Vazyme). RNA. The obtained RNA was reverse transcribed with polyT-containing primers to obtain cDNA.

2. PCR amplification

Use the cDNA obtained in step 1 as the template, use the primers P1 and P2 designed based on the TraesCS7A02G178300.1 transcript sequence as primers, and use high-fidelity enzymes to perform PCR amplification to obtain the PCR amplification product. The primer sequences are as follows:

Primer P1: 5’-CAGTAGCAAGCCATGGCCAA-3’;

Primer P2: 5’-GAACTGCACAATTAGCCGCTGC-3’.

The above PCR amplification reaction conditions: 95°C for 5 minutes; then 95°C for 15 seconds, 56°C for 15 seconds, 72°C for 1 minute, 32 cycles; and finally 72°C for 5 minutes.

3. Electrophoresis and sequencing

The PCR amplification product obtained in step 2 above was connected to the pMD-19T vector (TaKaRa) for sequencing. The sequencing results were spliced using DNAman software to obtain the full length of the TaGLP-7A gene, which was named TaGLP- 7A, the ORF sequence of the TaGLP-7A gene is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2.

Example 2: Expression analysis of TaGLP-7A in Bainong 207

1. Experimental method:

Take the seeds of Bainong 207, a common wheat material, and soak them in clean water at room temperature for 24 hours. After the seeds are swollen, pour out the liquid and keep them moist at room temperature for 24 hours. After they turn white, they are planted in a pot. Grow in a light incubator at 18°C/10h, 22°C/14h, and a humidity of 70%. Cultivate to the 2-leaf one-core stage and infect with fresh powdery mildew spores. Leaves of wheat seedlings infected for 0h, 1h, 24h, and 48h were quickly frozen in liquid nitrogen, and 3 plant leaves were cut at each time point. The above leaf materials were ground separately, total RNA was extracted, and reverse transcribed using HiScript III 1st Strand cDNA Synthesis Kit (+gDNAwiper) (Vazyme) to obtain cDNA. Using the cDNA as a template, primers P3 (CAACACCAGCAACCTCATCA) and Primer P4 (GGTGACGAAGAGGAGCTCAG) was used to amplify the TaGLP-7A fragment, and the TaTubulin internal reference primer was used to amplify the Tubulin fragment, and the expression of TaGLP-7A was analyzed.

The PCR procedure is as follows: PCR reaction is amplified on a real-time fluorescence quantitative PCR instrument (Roche, Germany) and fluorescence is detected. The 20uL PCR reaction system contains 10uL of 2×Taq Pro Universal SYBR qPCR Master Mix, 0.5μM primers P3 and P4, and 2uL of reverse transcription cDNA template. The amplification reaction conditions were: 95°C for 5 minutes, then 95°C for 15 seconds, and 60°C for 20 seconds, for a total of 40 cycles. After the reaction is completed, the melting curve is measured. Gene expression levels were detected and analyzed using sigmaplot14 software.

2. Experimental results

The detection results of real-time fluorescence quantitative PCR are shown in Figure 1. As can be seen from Figure 1, in Wheat Bainong 207, the expression of TaGLP-7A was significantly up-regulated 24 hours after being induced by powdery mildew, and returned to the original expression level after 48 hours. This shows that TaGLP-7A gene expression can respond to powdery mildew induction, and TaGLP-7A gene expression increases when powdery mildew infects.

Example 3: Obtaining silenced TaGLP-7A wheat and studying its powdery mildew resistance

1. Obtaining gene silencing fragments:

Using the pMD-18T vector containing the ORF sequence of TaGLP-7A as a template, PCR amplification was performed using primers P5 and P6 (P5 and P6 contain SmaI restriction sites and 15 bp complementary sequences to the vector), resulting in a fragment length of 248 bp. The target gene fragment, designated as TaGLP-7A (VIGS), was used to construct a VIGS silencing vector.

Among them, the nucleotide sequences of the above primers P5 and P6 are as follows:

P5: 5’-TAGCTGAGCGGCCGCCCCGGGGGAACACCATGATGTCGCCCT-3’;

P6: 5’-TAGCTGATTAATTAACCCGGGACACCAGCAACCTCATCAAGGC-3’.

The PCR amplification reaction system is: 1 μL plasmid template (30 ng/μL), 2 μL upstream primer P5, 2 μL downstream primer P6, 25 μL PCR mix, and add water to 50 μL.

PCR reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 20 s, 30 cycles, extension at 72°C for 5 min, and storage at 4°C. The PCR amplification products were recovered after detecting the amplified bands by 1.5% agarose gel electrophoresis.

2. Construction of BSMV recombinant viral vector:

According to conventional molecular biology methods, the silencing fragment obtained in step 1 above was inserted into the reverse direction between the Sma I restriction sites of the BSMV-VIGS viral vector γ through homologous recombination, while maintaining the BSMV- The other sequences of VIGS virus vector γ remained unchanged, and the recombinant vector γ-TaGLP-7A was obtained.

3. BSMV-VIGS vector system:

BSMV-VIGS viral vectors α, β and γ vectors together constitute the viral vector system BSMV:γ.

BSMV-VIGS viral vectors α, β and recombinant vector γ-TaGLP-7A together constitute the viral silencing vector system BSMV:γ-TaGLP-7A that can silence the TaGLP-7A gene.

BSMV-VIGS viral vectors α, β and vector γ-PDS together constitute the viral silencing vector system BSMV:γ-PDS that can silence the TaPDS gene. γ-PDS is from Scofield Laboratory (Scofield et al. 2005) and contains 185 bp. The 185 bp conserved fragment of the barley phytoene dehydrogenase gene (PDS) can be used as a positive control for gene silencing. After the gene is silenced, plant leaves appear whitening, which can be used to visually detect whether the silencing of the VIGS system is effective.

4. BSMV in vitro transcription:

(1)Linearization of carrier

The α chain, γ chain, recombinant vector γ-TaGLP-7A and recombinant vector γ-PDS of the BSMV viral vector were digested with MluI, and the β chain of the BSMV viral vector was digested with SpeI to obtain linearized plasmids respectively.

(2) Use the linearized plasmid obtained in the above step (1) as a template for in vitro transcription to obtain viral vectors α, β, γ, γ-TaGLP-7A and γ-PDS that are transcribed into RNA in vitro. The in vitro transcription reaction was performed according to the instructions of the mMESSAGEmMACHINE T7 in vitro transcription kit (Invitrogen). The transcription reaction system and conditions are: total reaction system 10.0 μL, including 3 μL linearized plasmid, 1 μL 10X Reaction Buffer, 5 μL 2x NTP/CAP, 1 μL Enzyme Mix, react in a PCR machine at 37°C for 2 hours, and heat the transcription product to -80°C Save for later use.

5. Wheat plant culture and BSMV inoculation

Select plump Bainong 207 seeds. After absorbing water in the petri dish for one day, pour out the water in the petri dish and rinse it clean. Keep the seeds moist for one day. After the seeds turn white, select wheat seeds with consistent growth and sow them in pots. After sowing, the wheat was grown in a light incubator with a temperature of 12°C/10°C and a lighting time of 14 hours of light/10 hours of darkness. Cultivate for a period of time until the wheat grows to two leaves with one center, and the second leaf is consistent with the first leaf, then select the wheat Bainong 207 with consistent growth. Before inoculating the virus, water the wheat enough, and apply the BSMV:γ-TaGLP-7A recombinant virus vector solution to the second leaf of the wheat using friction inoculation. The inoculated wheat was placed in an incubator at 23°C and kept in the dark for 24 hours. After 24 hours, the plants were converted to 23°C and grown under 14 hours of light/10 hours of darkness to obtain infected BSMV:γ-TaGLP-7A plants ( That is, wheat plants with silenced TaGLP-7A gene).

At the same time, some plants were inoculated with BSMV:γ-PDS virus vector solution to obtain BSMV:γ-PDS control plants, and some plants were inoculated with BSMV:γ virus vector solution to obtain BSMV:γ empty control plants.

The above-mentioned BSMV:γ-TaGLP-7A recombinant virus vector solution is made by mixing the in vitro transcription product with 10 μL α, 10 μL β, 10 μL γ-TaGLP-7A and 225 μL FES Buffer (0.1M glycine, 0.06MK ₂ HP0 ₄ buffer containing 1% sodium pyrophosphate, 1% macaloid , 1% celite; pH to 8.5-9.0 with phosphoricacid).

The above-mentioned BSMV:γ-PDS viral vector solution is a solution obtained by mixing the in vitro transcription product in a ratio of 10 μL α, 10 μL β, 10 μL γ-PDS and 225 μL FES Buffer.

The above BSMV:γ virus vector solution is obtained by mixing the in vitro transcription product in a ratio of 10 μL α, 10 μL β, 10 μL γ and 225 μL FE Buffer.

6. Identification of silenced TaGLP-7A wheat

In this experiment, about 14 days after applying the virus vector, the fourth leaf of the positive control wheat inoculated with the BSMV:γ-PDS virus vector solution showed a PDS albino phenotype, indicating that the genes in the wheat leaves at this period and corresponding leaf age had been silenced. This shows that the BSMV-VIGS system can be successfully applied to study the gene silencing function of wheat variety Bainong 207.

In order to further verify the effect of the BSMV-VIGS system in silencing the TaGLP-7A gene, its expression was detected using fluorescence quantitative PCR. The specific detection method is as follows: BSMV:γ-TaGLP-7A plants were infected with the smeared virus for 14 days. For BSMV:γ empty control plants, cut the fourth leaf with obvious symptoms of barley stripe mosaic virus to extract total RNA. After reverse transcription, the relative expression of TaGLP-7A gene was detected by real-time fluorescence quantitative PCR. Using TaTubulin as the internal reference gene, the relative expression was calculated by the 2 ^-ΔΔCt method. The primers used to detect expression levels are the same as those in Example 2.

The detection results of the relative expression level of TaGLP-7A gene are shown in Figure 2. As can be seen from Figure 2, the relative expression level of the TaGLP-7A gene in plants infected with BSMV:γ-TaGLP-7A is significantly lower than that in plants infected with BSMV:γ empty control, indicating that the silencing sequence selected in this experiment is Effective.

7. Analysis of powdery mildew resistance of silencing TaGLP-7A wheat

The leaf segments of the fourth leaf of the infected BSMV:γ-TaGLP-7A plants and the infected BSMV:γ empty control plants with the TaGLP-7A gene effectively silenced were placed on the 6-BA medium and inoculated with fresh white powder. The spores were cultured for 6 days at 22°C/18°C, 14h light/8h dark conditions. The phenotype was observed after 6 days, and the results are shown in Figure 3.

As can be seen from Figure 3, the disease incidence of control plants infected with BSMV:γ-empty fungus 6 days after inoculation with powdery mildew was significantly weaker than that of plants infected with BSMV:γ-TaGLP-7A. On the leaf surface of plants infected with BSMV:γ-TaGLP-7A A large number of spore clusters were produced, whereas the control leaves BSMV:γ produced a small number of spore clusters. It shows that silencing the TaGLP-7A gene in the common wheat Bainong 207 weakens the resistance of Bainong 207 to powdery mildew.

Example 4: Construction of TaGLP overexpression vector Pbi220:TaGLP-7A

Using the pMD-18T vector containing the ORF sequence of TaGLP-7A as a template, design primers P7 (GGAGAGAACACGGG GGATCC ATGGCCAACGCAATGCTGCTC, the underlined is the BamHI recognition base sequence) and P8 (AACGTCGTATTGGGTA AGGCCT GCCGCTGCCGCCGAGCA, the underlined is the StuI recognition base). base sequence) for PCR amplification; primer P7 has a BamHI restriction site, primer P8 has a StuI restriction site, and the PCR amplified fragments are recovered. The amplified product was inserted into the vector pBI220 double digested with BamHI and StuI by homologous recombination (Jefferson RA, Kavanagh TA, BevanMW. GUSfusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J.1987 , 6:3901-3907.), TaGLP-7A was placed at the multiple cloning site behind the 35S promoter. Thus, the target gene TaGLP-7A was cloned downstream of the strong promoter 35S, and the expression vector pBI220:TaGLP-7A was obtained (its structural diagram is shown in Figure 4). Sequencing verification showed that the vector was constructed successfully.

Example 5: Obtaining wheat plants overexpressing TaGLP-7A

The overexpressed pBI220:TaGLP-7A constructed in the above Example 4 was transferred into the seedling stage powdery mildew susceptible recipient Bainong 207 wheat callus using the gene gun transformation method to obtain overexpressed TaGLP-7A wheat plants.

The specific experimental steps are:

(1) Pick about 2,000 pieces of Bainong 207 embryonic callus pre-cultured for 7 days, and culture them in hypertonic medium (MS+ABA0.5mg/L+hydrolyzed casein 500mg/L+2, 4-D2mg/L+glucose 30g /L+0.4mol/L mannitol, pH5.8) for 4–5 hours;

(2) Co-transform the overexpression vector pBI220:TaGLP-7A carrying the target gene TaGLP-7A and the pAHC 25 vector carrying the bar marker into the Bainong 207 calli through gene gun bombardment, and then culture in hypertonic culture after bombardment Continue culturing on the base for 16 hours.

(3) Transfer the callus to recovery medium (1/2MS+hydrolyzed casein 500mg/L+2,4-D2mg/L+sucrose 30g/L, pH 5.8) and culture it in the dark for 2 weeks;

(4) Transfer the callus treated in step (3) to the screening medium containing herbicide (1/2MS+ABA0.5mg/L+hydrolyzed casein 500mg/L+2,4-D1mg/L+sucrose 30g/L+4mg/LBialaphos, pH5.8) screening and culture for 2 weeks;

(5) Transfer the herbicide-resistant calli screened in step (4) to differentiation medium (1/2MS+L-glutamine 1mmol/L+hydrolyzed casein 200mg/L+KT 1mg /L+IAA 0.5mg/L+sucrose 30g/L+agar 0.8%, pH5.8) for differentiation, and when the differentiated shoots grow to 2–4cm, transfer them to rooting medium (1/2MS+KT 1mg/L+sucrose 30g /L+agar 0.8%, pH5.8).

(6) When the regenerated seedlings are about 8cm long and the root system is relatively strong, the tubes can be opened and the seedlings can be hardened for 1-2 days. Finally, the medium residue carried by the roots can be washed away and then transplanted into pots. A total of 80 regenerated plants were obtained.

Genomic DNA of all regenerated plants was extracted, and PCR amplification was performed on the regenerated plants using promoter internal primer P9 (AGTGGAAAAGGAAGGTGGCT) and gene internal primer P10 (CATGATGTCGCCCTTGTAGAGC) to identify positive plants overexpressing TaGLP-7A.

The PCR reaction system is: 100ng genomic DNA template, 0.4μl each of 10μM P9 and P10; 5μl 2×mix; 3.2μl ddH ₂ O. PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes; 26 cycles of 95°C for 15 seconds, 59°C for 45 seconds, and 72°C for 50 seconds; extension at 72°C for 10 minutes. PCR products were detected by 1% agarose gel electrophoresis. After PCR amplification detection, three regenerated plants could amplify the target bands and were identified as positive plants (recorded as T ₀ generation positive plants). Figure 5 shows the screening of some positive plants of the T ₀ generation, including T ₀ -OE1, T ₀ -OE2, and T ₀ -OE3.

Example 6: Identification of powdery mildew resistance of wheat plants overexpressing TaGLP-7A

Harvest the seeds of the TaGLP-7A transgenic T0 generation positive plants T0-OE1, TO-OE2, and TO-OE3 obtained through screening in Example 5 respectively, and use the harvested seeds of the T0 generation positive plants T0-OE1, TO-OE2, and TO-OE3. The seeds were planted in pots to obtain T ₁ -OE1 plants, T ₁ -OE2 plants and T ₁ -OE3 plants. At the two-leaf and one-heart stage, RNA was extracted from the three identified positive lines for qRT-PCR analysis. The specific experimental methods were the same as in Example 2. The results showed that the TaGLP-7A positive lines T ₁ -OE1 plant and T ₁ -OE2 plant were found. and the expression level of T ₁ -OE3 plants was significantly higher than the negative control T1-42 (Fig. 6). At the three-leaf stage, the T ₁ -OE1 plant, T ₁ -OE2 plant and T ₁ -OE3 plant were cut off the leaf segments of the third leaf at the three-leaf one-heart stage and placed on the 6-BA medium. At the same time, the susceptible receptors were Bainong 207 and transgenic negative plants were used as negative controls, and were inoculated with fresh powdery mildew spores and cultured for 6 days at 22°C/18°C, 14h light/8h dark conditions. Six days after inoculation of powdery mildew spores, the phenotype was observed, and the results are shown in Figure 7.

It can be seen from Figure 7 that the susceptible control plant Bainong 207 and the transgenic negative control plant are highly susceptible to powdery mildew, and the leaves are covered with powdery mildew spore piles; T ₁ -OE1 plant, T ₁ -OE2 plant and T ₁ -OE3 plant There are only a few powdery mildew spores on the leaves. This shows that compared with the untransformed Bainong 207 and the regenerated negative lines, the powdery mildew resistance level of the transgenic T ₁ generation positive plants (T ₁ -OE1 plants, T ₁ -OE2 plants and T ₁ -OE3 plants) has been significantly improved. Improved, T ₁ -OE1 plant, T ₁ -OE2 plant and T ₁ -OE3 plant all showed disease resistance.

The above identification results show that increasing the expression level of TaGLP-7A in susceptible wheat plants can significantly improve the powdery mildew resistance of susceptible wheat plants. Therefore, this gene can be used to breed powdery mildew-resistant wheat using genetic engineering means.

The above are only preferred embodiments of the present invention, but are not limited to the above examples. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. within.

sequence list

<110> Henan University of Science and Technology

<120> Wheat powdery mildew resistance-related protein TaGLP-7A and its encoding genes and applications

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<170> SIPOSequenceListing 1.0

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tacccgtgca agaccggcgt cggcgcgggg gacttctact accacggcct cgccgccgcg 180

ggcaacacca gcaacctcat caaggcggcc gtgaccccgg ccttcgtcgg ccagttcccc 240

ggcgtgaacg ggctcggcat ctccgcggcg aggctcgaca tcgccgtggg cggcgtcgtg 300

ccgctgcaca cccacccggc cgcctctgag ctcctcttcg tcaccgaggg caccatcctg 360

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atcatggtgt tcccccaggg cctgctccac taccagtaca acggcggcgg ctcggcagcg 480

gtggcgctcg ttgcgttcag cggccccaac cccggcctgc agatcactga ctacgcgctc 540

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Met Ala Asn Ala Met Leu Leu Pro Val Leu Ile Ser Phe Leu Val Leu

1 5 10 15

Pro Phe Ser Ala Leu Ala Leu Thr Gln Asp Phe Cys Val Ala Asp Leu

20 25 30

Ser Cys Ser Asp Thr Pro Ala Gly Tyr Pro Cys Lys Thr Gly Val Gly

35 40 45

Ala Gly Asp Phe Tyr Tyr His Gly Leu Ala Ala Ala Gly Asn Thr Ser

50 55 60

Asn Leu Ile Lys Ala Ala Val Thr Pro Ala Phe Val Gly Gln Phe Pro

65 70 75 80

Gly Val Asn Gly Leu Gly Ile Ser Ala Ala Arg Leu Asp Ile Ala Val

85 90 95

Gly Gly Val Val Pro Leu His Thr His Pro Ala Ala Ser Glu Leu Leu

100 105 110

Phe Val Thr Glu Gly Thr Ile Leu Ala Gly Phe Ile Ser Ser Ser Ser

115 120 125

Asn Thr Val Tyr Thr Lys Thr Leu Tyr Lys Gly Asp Ile Met Val Phe

130 135 140

Pro Gln Gly Leu Leu His Tyr Gln Tyr Asn Gly Gly Gly Ser Ala Ala

145 150 155 160

Val Ala Leu Val Ala Phe Ser Gly Pro Asn Pro Gly Leu Gln Ile Thr

165 170 175

Asp Tyr Ala Leu Phe Ala Asn Asn Leu Pro Ser Ala Val Val Glu Lys

180 185 190

Val Thr Phe Leu Asp Asp Ala Gln Val Lys Lys Leu Lys Ser Val Leu

195 200 205

Gly Gly Ser Gly

210

Claims (4)

1. The application of the protein with the amino acid sequence shown as SEQ ID NO.2 or the nucleic acid molecule encoding the protein or the recombinant vector containing the nucleic acid molecule, the expression cassette or the recombinant bacteria in improving the powdery mildew resistance of plants;

or, the application of the protein with the amino acid sequence shown as SEQ ID NO.2 or the nucleic acid molecule encoding the protein or the recombinant vector, the expression cassette or the recombinant bacteria containing the nucleic acid molecule in cultivating transgenic plants with improved powdery mildew resistance;

wherein the sequence of the nucleic acid molecule is shown as SEQ ID NO.1, and the plant is wheat.

2. A method of growing a transgenic plant with increased powdery mildew resistance comprising the step of increasing the expression and/or activity of a protein in a recipient plant to obtain a transgenic plant; the transgenic plant has a powdery mildew resistance higher than the recipient plant; the amino acid sequence of the protein is shown as SEQ ID NO.2, and the plant is wheat.

3. The method of claim 2, wherein the method of increasing the expression and/or activity of the protein in the recipient plant is: the protein is overexpressed in the recipient plant.

4. A method according to claim 3, wherein the over-expression is by introducing a gene encoding the protein into a recipient plant.