A wheat aphid-resistant gene TaAHL19, its application, and a method for obtaining aphid-resistant wheat varieties.

Abstract

This invention provides a wheat aphid-resistant gene TaAHL19, its application, and a method for obtaining aphid-resistant wheat varieties. The nucleotide sequence of the wheat aphid-resistant gene TaAHL19 is shown in SEQ ID NO.1. The amino acid sequence encoded by the wheat aphid-resistant gene TaAHL19 is shown in SEQ ID NO.2. In this invention, overexpression of the TaAHL19 gene in wheat plants promotes the accumulation of rutin, an insect-resistant flavonoid, in wheat plants, thereby increasing the resistance of wheat plants to aphids without affecting wheat yield.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a wheat aphid-resistant gene TaAHL19, its application, and a method for obtaining aphid-resistant wheat varieties. Background Technology

[0002] Wheat is an important staple food crop in my country, and the wheat aphid is one of the most serious pests causing wheat losses. Currently, wheat aphid control mainly relies on chemical control, but the irrational use of chemical pesticides has led to increased pesticide resistance in pests, threatening human and animal health and environmental safety. Therefore, research on green control technologies for wheat aphids to reduce the use of chemical pesticides and improve ecosystem benefits is urgently needed. Breeding and utilizing aphid-resistant varieties is one of the most economical and effective strategies for the sustainable control of wheat aphids. However, currently, wheat varieties suitable for large-scale application are relatively scarce, and high-quality aphid-resistant gene pools are relatively lacking. Therefore, using modern biotechnology to discover superior insect-resistant genes and analyze their molecular mechanisms is of great significance for the development of high-quality aphid-resistant wheat varieties.

[0003] Through long-term co-evolution, host plants and pests have developed various insect resistance mechanisms. Nutrients and toxic secondary metabolites in wheat can directly affect the growth and development of wheat aphids, thus influencing wheat's aphid resistance. As products of metabolic pathways, most wheat secondary metabolites enhance wheat's resistance to pests and diseases, making them an important class of insect-resistant defense substances. Flavonoids are an important class of secondary metabolites related to plant resistance, possessing strong physiological activity. Flavonoids are typically induced after damage by herbivorous insects, inhibiting their feeding and growth. Rutin, a flavonol secondary metabolite, enhances plant stress tolerance, inhibits bacteria, provides antioxidant effects, and scavenges free radicals, playing a crucial role in plant resistance to pathogens and insect damage. For example, the Arabidopsis thaliana AtMYB12 gene regulates flavonoid synthesis, leading to a large accumulation of rutin. Overexpression of the AtMYB12 gene in tobacco enhances the plant's resistance to aphids and whiteflies.

[0004] Research on wheat aphid resistance genes has, to date, identified 15 aphid resistance genes (Gb), of which 2 alleles originate from wheat and 10 alleles originate from jointed goatgrass. Aegilops tauschii One comes from *Aegilops spp.* A. speltoides Two of them are from rye. Secale cereale Currently, there is limited research on resistance genes and genetics of the wheat aphid. Furthermore, high-quality aphid-resistant gene banks are relatively scarce, and wheat varieties suitable for large-scale application are relatively limited. Therefore, it is necessary to further enrich the aphid-resistant gene bank and identify aphid-resistant genes that can enhance wheat plants' resistance to aphids without affecting wheat yield, for use in the creation of aphid-resistant wheat varieties. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a wheat aphid-resistant gene TaAHL19, its application, and a method for obtaining aphid-resistant wheat varieties. Overexpression of the TaAHL19 gene in wheat plants can promote the accumulation of rutin, an insect-resistant flavonoid, in wheat plants, thereby increasing the resistance of wheat plants to aphids without affecting wheat yield.

[0006] To address the aforementioned problems, a first aspect of the present invention provides a wheat aphid-resistant gene TaAHL19, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] A second aspect of the present invention provides an amino acid sequence encoded by the above-mentioned wheat aphid-resistant gene TaAHL19, the amino acid sequence being shown in SEQ ID NO.2.

[0008] A third aspect of the present invention provides a recombinant vector or host bacterium containing the aforementioned wheat aphid-resistant gene TaAHL19.

[0009] The fourth aspect of the present invention provides the application of the above-mentioned wheat aphid-resistant gene TaAHL19 or the above-mentioned amino acid sequence, by overexpressing the wheat aphid-resistant gene TaAHL19, to improve wheat aphid resistance or increase the rutin content in wheat plants.

[0010] A fifth aspect of the present invention provides a method for improving wheat aphid resistance, comprising the step of overexpressing the wheat aphid resistance gene TaAHL19 in wheat plants; the nucleotide sequence of the wheat aphid resistance gene TaAHL19 is shown in SEQ ID NO.1.

[0011] A sixth aspect of the present invention provides a method for obtaining an aphid-resistant wheat variety, comprising the following steps: S1. The wheat aphid resistance gene TaAHL19 was inserted into a vector to construct an overexpression vector; the nucleotide sequence of the wheat aphid resistance gene TaAHL19 is shown in SEQ ID NO.1; S2. The overexpression vector is introduced into Agrobacterium to obtain an engineered Agrobacterium carrying the overexpression vector; S3. Using Agrobacterium-mediated transformation of immature wheat embryos carrying an overexpression vector, the wheat aphid-resistant gene TaAHL19 was integrated into the wheat genome, resulting in wheat plants overexpressing the TaAHL19 gene.

[0012] Preferably, step S1 specifically includes the following steps: The full-length coding region of the TaAHL19 gene was amplified and ligated into the cloning vector pTOPO-TA / Blunt. The resulting recombinant cloning vector TaAHL19-T was transformed into E. coli DH5α competent cells using the heat shock method. Positive clones were cultured and plasmids were extracted using a plasmid mini-DNA extraction kit. Using the extracted plasmid as a template, TaAHL19 was inserted between the EcoRI and BamHI restriction sites of the pWMB111 vector to construct the pWMB111-TaAHL19 overexpression vector via homologous recombination.

[0013] Preferably, in step S1, when amplifying the full-length coding region of the TaAHL19 gene, the PCR primers used include a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO.3; and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4.

[0014] Preferably, step S2 specifically includes the following steps: Under aseptic conditions, recombinant plasmid pWMB111-TaAHL19 DNA was added to Agrobacterium EHA105 competent cells, mixed well, and incubated on ice for 5 min. Then, the cells were rapidly frozen in liquid nitrogen for 5 min, quickly placed in a 37°C metal bath for 5 min, and then returned to an ice-water bath for 5 min. Under aseptic conditions, LB liquid medium was added, and the cells were cultured at 28°C and 200 rpm for 3 h with shaking to allow the cells to recover. The cells were collected by centrifugation at 5000 rpm for 1 min, and part of the supernatant was discarded. 150 μL of the cells were resuspended by pipetting and spread on LB solid medium containing Kana and Rif. The cells were incubated upside down at 28°C for 2-3 days to obtain the Agrobacterium EHA105 engineered strain carrying the recombinant plasmid pWMB111-TaAHL19.

[0015] Preferably, step S3 specifically includes the following steps: Wheat ears were collected, and immature grains were gathered. Under aseptic conditions, the grains were surface-sterilized with ethanol, then with sodium hypochlorite, and finally rinsed with sterile water to obtain sterilized immature wheat embryos. The sterilized immature wheat embryos were transformed using Agrobacterium EHA105 engineered strain carrying the recombinant plasmid pWMB111-TaAHL19. The transformed tissues were transferred to callus induction medium supplemented with 5 mg / L phosphinic acid PPT to further induce callus formation. The regenerated shoots were then transplanted into culture cups containing rooting medium containing 5 mg / L PPT to promote shoot elongation and rooting. Seedlings with well-developed root systems were transplanted into flower pots and cultured in a growth chamber to obtain wheat plants overexpressing the TaAHL19 gene. Compared with the prior art, the present invention has the following advantages: This invention, through research on aphid-resistant wheat varieties, identified and precisely located a gene, TaAHL19, that enhances wheat's resistance to aphids. Overexpression of the TaAHL19 gene in wheat plants promotes the accumulation of rutin, an insect-resistant flavonoid, and significantly improves wheat's resistance to wheat aphids. Overexpression of this wheat aphid-resistant gene, TaAHL19, in wheat enhances aphid resistance without affecting wheat yield, enriching the aphid-resistant gene pool and providing new candidate genes for the creation of new aphid-resistant germplasm. Attached Figure Description

[0016] Figure 1 This is the agarose gel electrophoresis identification result of the TaAHL19 gene overexpressing plants in Example 1 of this invention; Figure 2 This refers to the relative expression level of the TaAHL19 gene in the TaAHL19 gene overexpressing plants in Example 1 of this invention; where WT represents wild-type wheat plants; OE2 represents TaAHL19-OE2; and OE6 represents TaAHL19-OE6. Figure 3 This describes the effect of TaAHL19 gene overexpression on grain length in Example 1 of this invention; where WT represents wild-type wheat plants; OE2 represents TaAHL19-OE2; and OE6 represents TaAHL19-OE6. Figure 4 This describes the effect of TaAHL19 gene overexpression on grain width in Example 1 of the present invention; where WT represents wild-type wheat plants; OE2 represents TaAHL19-OE2; and OE6 represents TaAHL19-OE6. Figure 5 This describes the effect of TaAHL19 gene overexpression on the thousand-grain weight in Example 1 of this invention; where WT represents wild-type wheat plants; OE2 represents TaAHL19-OE2; and OE6 represents TaAHL19-OE6. Figure 6 This is the effect of TaAHL19 gene overexpression on rutin content in wheat plants in Example 2 of the present invention; wherein, WT is wild-type wheat plant; OE2 is TaAHL19-OE2; Figure 7 This is the effect of TaAHL19 gene overexpression on the aphid production of wheat aphid in Example 3 of the present invention; where WT is a wild-type wheat plant; and OE2 is TaAHL19-OE2. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] A first aspect of the present invention provides a wheat aphid-resistant gene TaAHL19, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0019] SEQ ID NO.1: .

[0020] This invention, through research on aphid-resistant wheat varieties, identified and precisely located a gene, TaAHL19, that enhances wheat's resistance to aphids. Overexpression of the TaAHL19 gene in wheat plants promotes the accumulation of rutin, an insect-resistant flavonoid, and significantly improves wheat's resistance to wheat aphids. Overexpression of this wheat aphid-resistant gene, TaAHL19, in wheat enhances aphid resistance without affecting wheat yield, enriching the aphid-resistant gene pool and providing new candidate genes for the creation of new aphid-resistant germplasm.

[0021] A second aspect of the present invention provides an amino acid sequence encoded by the above-mentioned wheat aphid-resistant gene TaAHL19, the amino acid sequence being shown in SEQ ID NO.2.

[0022] SEQ ID NO.2: MGSMDGHPLQGNHAYAHVPAGSNNDEDDASPPPSAGGGSGSGRRPRGRPPGSKNKPKPPVVVTRESPNAMRSHVLEIASGADIVEAIAAFSRRRQRGVSVLSGSGAVTNVTLRQPAGTGAAAVALRG RFEILSLSGAFLPAPAPPGATGLAVYLAGGQGQVVGGSVMGELLASGPVMVIAATFGNATYERLPLDQDAEEGAVLSGSEGAATQLEQQGSGGAAVPPPMYAVPQTPPSDVFGQWGQAAVARPPPTSF.

[0023] A third aspect of the present invention provides a recombinant vector or host bacterium containing the aforementioned wheat aphid-resistant gene TaAHL19.

[0024] The fourth aspect of the present invention provides the application of the above-mentioned wheat aphid-resistant gene TaAHL19 or the above-mentioned amino acid sequence, by overexpressing the wheat aphid-resistant gene TaAHL19, to improve wheat aphid resistance or increase the rutin content in wheat plants.

[0025] Preferably, the aphid resistance of wheat is the aphid resistance of wheat to the wheat long-tubed aphid.

[0026] A fifth aspect of the present invention provides a method for improving wheat aphid resistance, comprising the step of overexpressing the wheat aphid resistance gene TaAHL19 in wheat plants; the nucleotide sequence of the wheat aphid resistance gene TaAHL19 is shown in SEQ ID NO.1.

[0027] A sixth aspect of the present invention provides a method for obtaining an aphid-resistant wheat variety, comprising the following steps: S1. The wheat aphid resistance gene TaAHL19 was inserted into a vector to construct an overexpression vector; the nucleotide sequence of the wheat aphid resistance gene TaAHL19 is shown in SEQ ID NO.1; S2. The overexpression vector is introduced into Agrobacterium to obtain an engineered Agrobacterium carrying the overexpression vector; S3. Using Agrobacterium-mediated transformation of immature wheat embryos carrying an overexpression vector, the wheat aphid-resistant gene TaAHL19 was integrated into the wheat genome, resulting in wheat plants overexpressing the TaAHL19 gene.

[0028] Preferably, step S1 specifically includes the following steps: The full-length coding region of the TaAHL19 gene was amplified and ligated into the cloning vector pTOPO-TA / Blunt. The resulting recombinant cloning vector TaAHL19-T was transformed into E. coli DH5α competent cells using the heat shock method. Positive clones were cultured and plasmids were extracted using a plasmid mini-DNA extraction kit. Using the extracted plasmid as a template, TaAHL19 was inserted between the EcoRI and BamHI restriction sites of the pWMB111 vector to construct the pWMB111-TaAHL19 overexpression vector via homologous recombination.

[0029] Preferably, in step S1, when amplifying the full-length coding region of the TaAHL19 gene, the PCR primers used include a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO.3; and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4.

[0030] SEQ ID NO. 3: ATGGGGAGCATGGACGGCCACCCGCT.

[0031] SEQ ID NO. 4: CTAGAATGACGTCGGCGGAGGCCG.

[0032] Preferably, step S2 specifically includes the following steps: Under aseptic conditions, recombinant plasmid pWMB111-TaAHL19 DNA was added to Agrobacterium EHA105 competent cells, mixed well, and incubated on ice for 5 min. Then, the cells were rapidly frozen in liquid nitrogen for 5 min, quickly placed in a 37°C metal bath for 5 min, and then returned to an ice-water bath for 5 min. Under aseptic conditions, LB liquid medium was added, and the cells were cultured at 28°C and 200 rpm for 3 h with shaking to allow the cells to recover. The cells were collected by centrifugation at 5000 rpm for 1 min, and part of the supernatant was discarded. 150 μL of the cells were resuspended by pipetting and spread on LB solid medium containing Kana and Rif. The cells were incubated upside down at 28°C for 2-3 days to obtain the Agrobacterium EHA105 engineered strain carrying the recombinant plasmid pWMB111-TaAHL19.

[0033] Preferably, step S3 specifically includes the following steps: Wheat ears were collected, and immature grains were gathered. Under aseptic conditions, the grains were surface-sterilized with ethanol, then with sodium hypochlorite, and finally rinsed with sterile water to obtain sterilized immature wheat embryos. The sterilized immature wheat embryos were transformed using Agrobacterium EHA105 engineered strain carrying the recombinant plasmid pWMB111-TaAHL19. The transformed tissues were transferred to callus induction medium supplemented with 5 mg / L phosphinic acid PPT to further induce callus formation. The regenerated shoots were then transplanted into culture cups containing rooting medium containing 5 mg / L PPT to promote shoot elongation and rooting. Seedlings with well-developed root systems were transplanted into flower pots and cultured in a growth chamber to obtain wheat plants overexpressing the TaAHL19 gene.

[0034] Example 1: Construction of wheat lines overexpressing the TaAHL19 gene 1. Synthesis and Sequence Determination of the TaAHL19 Gene The nucleotide sequence (771 nucleotides) of the wheat TaAHL19 gene is shown in SEQ ID NO.1 of the sequence listing, and the encoded amino acid sequence (256 amino acids, with the last 3 nucleotides being the stop codon TAG, which is not translated into amino acids) is shown in SEQ ID NO.2 of the sequence listing. The full-length coding region of the TaAHL19 gene was amplified and ligated into the cloning vector pTOPO-TA / Blunt (Aidlab, Beijing, China). The recombinant cloning vector TaAHL19-T was transformed into *E. coli* DH5α competent cells (Biomed, Beijing, China) using the heat shock method. Positive clones were cultured and plasmids were extracted using a plasmid mini-DNA extraction kit (Axygen, California, America). Sequencing of the extracted plasmids confirmed that the TaAHL19 nucleotide sequence inserted into the recombinant cloning vector TaAHL19-T was the same as the nucleotide sequence shown in SEQ ID NO.1 of the sequence listing, indicating that the TaAHL19 nucleotide sequence was correctly inserted.

[0035] PCR primer sequences. The nucleotide sequence of the forward primer is shown in SEQ ID NO.3; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4.

[0036] SEQ ID NO. 3: ATGGGGAGCATGGACGGCCACCCGCT.

[0037] SEQ ID NO. 4: CTAGAATGACGTCGGCGGAGGCCG.

[0038] 2. Carrier Construction Using the plasmids described above as templates, TaAHL19 was inserted between the EcoRI and BamHI restriction sites of the pWMB111 vector (which contains a Bar expression cassette controlled by the ZmUbi promoter) to construct the pWMB111-TaAHL19 overexpression vector via homologous recombination.

[0039] 3. Genetic transformation The pWMB111-TaAHL19 overexpression vector was transformed into Agrobacterium EHA105 strain. The specific steps were as follows: Under aseptic conditions, 5 μL of recombinant plasmid DNA was added to competent cells, gently mixed, and incubated on ice for 5 min; the centrifuge tube was rapidly frozen in liquid nitrogen for 5 min, then quickly placed in a 37℃ metal bath for 5 min, and finally returned to an ice-water bath for 5 min; under aseptic conditions, 800 μL of LB liquid medium equilibrated to room temperature was added, and the cells were incubated at 28℃ and 200 rpm with shaking for 3 h to allow the cells to recover; the cells were collected by centrifugation at 5000 rpm for 1 min, some supernatant was discarded, and 150 μL of the cells were gently resuspended by pipetting. The cells were then spread onto LB solid medium containing Kana (50 μg / mL) and Rif (20 μg / mL) and incubated upside down at 28℃ for 2-3 days.

[0040] Wheat ears were harvested 14 days after flowering, and immature grains were carefully collected. Under aseptic conditions, the grains were surface-sterilized with 70% ethanol for 1 minute, followed by sterilization with 5% sodium hypochlorite for 15 minutes, and then rinsed 5 times with sterile water. Fresh immature embryos were isolated for Agrobacterium-mediated transformation. The tissues were transferred to a selective medium (callus induction medium supplemented with 5 mg / L phosphinic acid PPT) to further induce callus formation. Regenerated shoots were then transplanted into culture cups containing rooting medium containing 5 mg / L PPT to promote shoot elongation and rooting. Seedlings with well-developed root systems were transplanted into pots and cultured in a growth chamber.

[0041] 4. Detection of positive plants and detection of TaAHL19 gene expression level To detect positive plants, leaf DNA was extracted from 15 TaAHL19 overexpressing lines and amplified by PCR. Figure 1 As shown, 14 out of 15 overexpression lines were positive, indicating that wheat overexpressing the TaAHL19 gene was successfully constructed. The TaAHL19-OE2 and TaAHL19-OE6 lines were selected for TaAHL19 gene expression level detection and subsequent aphid resistance analysis.

[0042] The PCR primer sequences for detecting positive plants are shown in SEQ ID NO.5 for the forward primer and SEQ ID NO.6 for the reverse primer.

[0043] SEQ ID NO. 5: TTTAGCCCTGCCTCATACGCT.

[0044] SEQ ID NO. 6: TGTATAATTGCGGGACTCTAATC.

[0045] RNA was extracted from leaves of wheat lines TaAHL19-OE2 and TaAHL19-OE6 using the Trizol method, reverse transcribed into cDNA, and the expression level of the TaAHL19 gene was detected at the mRNA level using real-time quantitative PCR. Fielder wild-type wheat plants were used as controls.

[0046] The nucleotide sequences of the quantitative PCR primers for TaAHL19 are shown in SEQ ID NO.7 for the forward primer and in SEQ ID NO.8 for the reverse primer.

[0047] SEQ ID NO.7: ATGGGGAGCATGGACG.

[0048] SEQ ID NO. 8: CTTGGGCTTGTTCTTG.

[0049] The internal reference gene is TaActin, and the nucleotide sequence of its quantitative PCR primers is shown in SEQ ID NO.9 for the forward primer and in SEQ ID NO.10 for the reverse primer.

[0050] SEQ ID NO.9: GGAAAATCAGTCTCGGTTCAG.

[0051] SEQ ID NO. 10: TCATACAGCAGGCAAGCAC.

[0052] like Figure 2 As shown, the expression level of the TaAHL19 gene in both overexpression lines was significantly upregulated compared to wild-type wheat plants, with TaAHL19-OE2 and TaAHL19-OE6 showing upregulation rates of 37.23% and 11.07%, respectively. The TaAHL19-OE2 line, with the highest upregulation rate, was selected for subsequent aphid resistance assessment and rutin content detection.

[0053] 5. Detection of agronomic traits in plants overexpressing the TaAHL19 gene and wild-type plants Ten grains from TaAHL19-OE2, TaAHL19-OE6, and Fielder wild-type wheat lines were randomly selected, and the grain length and width were measured using vernier calipers. The thousand-grain weight of the three lines (TaAHL19-OE2, TaAHL19-OE6, and Fielder) was also measured.

[0054] The results showed that TaAHL19-OE2, TaAHL19-OE6 and wild-type wheat had no significant differences in grain length, grain width, and thousand-grain weight. Figure 3 , Figure 4 , Figure 5 This indicates that TaAHL19 gene overexpression has no significant effect on wheat yield.

[0055] Example 2: Effect of TaAHL19 gene overexpression on rutin content in wheat plants Wheat seeds (TaAHL19-OE2 and Fielder wild-type wheat) were sown in potting soil and cultured in a greenhouse with a relative humidity of 65 ± 5%, a temperature of 20 ± 1℃, and a light intensity (L:D) of 16 h:8 h. Wheat leaves from the wild-type wheat and the TaAHL19-OE2 line were harvested after 12 days. Each treatment had four technical replicates.

[0056] Accurately weigh approximately 0.1 g of dried and sieved wheat leaf sample, add 1 mL of methanol, and extract four times. Concentrate the extract by rotary evaporation to 0.2 mL, and filter the supernatant through a 0.45 μm filter membrane for use as the test solution. The rutin content was determined using high-performance liquid chromatography (HPLC). A WD-C18 (250 mm x 4.6 mm, 5 μm) column was used; the flow rate was 600 μL / min; the injection volume was 10 μL; and the mobile phase was methanol:water (containing 0.4% phosphoric acid) = 45:55 (V:V). The rutin content was analyzed based on the rutin standard curve.

[0057] The results of the rutin content analysis are as follows: Figure 6 As shown, TaAHL19-OE wheat plants had a significantly increased rutin content compared to wild-type wheat plants. This indicates that overexpression of the TaAHL19 gene significantly increases the rutin content in wheat plants.

[0058] Example 3: Effects of TaAHL19 gene overexpression on aphid resistance in wheat lines The planting method for wild-type wheat and TaAHL19-OE2 plants was the same as in Example 2. Wheat grown to 12 days old was used for aphid resistance identification tests.

[0059] The wheat aphid population tested was collected from wheat fields at the Langfang Research and Pilot Base of the Chinese Academy of Agricultural Sciences. It was raised year-round on potted wheat (aphid-susceptible variety Aikang 58) in the greenhouse of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The rearing conditions were: relative humidity 65 ± 5%, temperature 20 ± 1℃, and light intensity L:D = 16h:8h.

[0060] Two wingless adult aphids were introduced onto each wheat seedling. After 24 hours, the adult aphids and any excess nymphs were removed, leaving one first-instar nymph per seedling. The aphid count was recorded after 10 days. Each treatment had 18 technical replicates.

[0061] The results are as follows Figure 7 As shown, the number of wheat aphids on TaAHL19-OE2 plants was significantly reduced compared to wild-type wheat plants. This indicates that overexpression of the TaAHL19 gene can significantly improve the aphid resistance of wheat.

[0062] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A wheat aphid-resistant gene TaAHL19, characterized by: The nucleotide sequence of the wheat aphid-resistant gene TaAHL19 is shown in SEQ ID NO.

1.

2. An amino acid sequence encoded by the wheat aphid-resistant gene TaAHL19 as described in claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant vector or host bacterium containing the wheat aphid-resistant gene TaAHL19 as described in claim 1.

4. The application of the wheat aphid-resistant gene TaAHL19 as described in claim 1 or the amino acid sequence as described in claim 2, characterized in that, Overexpression of the wheat aphid-resistant gene TaAHL19 can enhance wheat aphid resistance or increase the rutin content in wheat plants.

5. A method for improving wheat's resistance to aphids, characterized in that: The method includes the step of overexpressing the wheat aphid-resistant gene TaAHL19 in wheat plants; the nucleotide sequence of the wheat aphid-resistant gene TaAHL19 is shown in SEQ ID NO.

1.

6. A method for obtaining aphid-resistant wheat varieties, characterized in that, Includes the following steps: S1. The wheat aphid resistance gene TaAHL19 was inserted into a vector to construct an overexpression vector; the nucleotide sequence of the wheat aphid resistance gene TaAHL19 is shown in SEQ ID NO.1; S2. The overexpression vector is introduced into Agrobacterium to obtain an engineered Agrobacterium carrying the overexpression vector; S3. Using Agrobacterium-mediated transformation of immature wheat embryos carrying an overexpression vector, the wheat aphid-resistant gene TaAHL19 was integrated into the wheat genome, resulting in wheat plants overexpressing the TaAHL19 gene.

7. The method for obtaining aphid-resistant wheat varieties according to claim 6, characterized in that, Step S1 specifically includes the following steps: The full-length coding region of the TaAHL19 gene was amplified and ligated into the cloning vector pTOPO-TA / Blunt. The resulting recombinant cloning vector TaAHL19-T was transformed into E. coli DH5α competent cells using the heat shock method. Positive clones were cultured and plasmids were extracted using a plasmid mini-DNA extraction kit. Using the extracted plasmid as a template, TaAHL19 was inserted between the EcoRI and BamHI restriction sites of the pWMB111 vector to construct the pWMB111-TaAHL19 overexpression vector via homologous recombination.

8. The method for obtaining aphid-resistant wheat varieties according to claim 7, characterized in that: In step S1, when amplifying the full-length coding region of the TaAHL19 gene, the PCR primers used include a forward primer and a reverse primer; the nucleotide sequence of the forward primer is shown in SEQ ID NO.3; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

4.

9. The method for obtaining aphid-resistant wheat varieties according to claim 7, characterized in that, Step S2 specifically includes the following steps: Under aseptic conditions, recombinant plasmid pWMB111-TaAHL19 DNA was added to Agrobacterium EHA105 competent cells, mixed well, and incubated on ice for 5 min. Then, the cells were rapidly frozen in liquid nitrogen for 5 min, quickly placed in a 37°C metal bath for 5 min, and then returned to an ice-water bath for 5 min. Under aseptic conditions, LB liquid medium was added, and the cells were cultured at 28°C and 200 rpm for 3 h to allow the cells to recover. The cells were collected by centrifugation at 5000 rpm for 1 min, and part of the supernatant was discarded. 150 μL of the cells were resuspended by pipetting and spread on LB solid medium containing Kana and Rif. The cells were incubated upside down at 28°C for 2-3 days to obtain the Agrobacterium EHA105 engineered strain carrying the recombinant plasmid pWMB111-TaAHL19.

10. The method for obtaining aphid-resistant wheat varieties according to claim 7, characterized in that, Step S3 specifically includes the following steps: Wheat ears were collected, and immature grains were gathered. Under aseptic conditions, the grains were surface-sterilized with ethanol, then with sodium hypochlorite, and finally rinsed with sterile water to obtain sterilized immature wheat embryos. The sterilized immature wheat embryos were transformed using Agrobacterium EHA105 engineered strain carrying the recombinant plasmid pWMB111-TaAHL19. The transformed tissues were transferred to callus induction medium supplemented with 5 mg / L phosphinic acid PPT to further induce callus formation. The regenerated shoots were then transplanted into culture cups containing rooting medium containing 5 mg / L PPT to promote shoot elongation and rooting. Seedlings with well-developed root systems were transplanted into flower pots and cultured in a growth chamber to obtain wheat plants overexpressing the TaAHL19 gene.

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