Application of TaACT4-3 gene in regulation and control of plant phenol amine substance content and / or plant powdery mildew resistance

Abstract

The invention belongs to the technical field of gene engineering and plant disease prevention and control, and particularly relates to application of a TaACT4-3 gene to regulation and control of plant phenol amine substance content and / or plant powdery mildew resistance. The invention provides an application of a TaACT4-3 gene in regulating and controlling the content of plant phenol amine substances and / or regulating and controlling the powdery mildew resistance of plants. The amino acid sequence of the protein coded by the TaACT4-3 gene is as shown in SEQ ID NO: 3. The TaACT4-3 gene is developed and utilized, and the TaACT4-3 gene is over-expressed, so that the lignin content and the hydrogen peroxide content of the plant can be increased, the content of phenol amine substances in the plant, especially wheat, is effectively increased, the resistance of the plant to powdery mildew is enhanced, and the original quantitative character of the plant is not influenced.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and plant disease prevention and control, and specifically relates to the application of TaACT4-3 gene in regulating the content of plant phenolamine substances and / or plant powdery mildew resistance. Background Art

[0002] Wheat powdery mildew caused by Blumeria graminis f.sp.Tritici (Bgt) is a global fungal disease. The infection of wheat by powdery mildew causes leaf death, and reduces the number of tillers, ear setting rate, number of grains per ear, and thousand-grain weight, which seriously threatens the yield and quality of wheat. At present, the prevention and control of wheat powdery mildew in production is still mainly based on the spraying of chemical agents and reasonable dense planting. However, the use of chemical agents such as triadimenol and triadimefon will not only increase the cost of field input, but also the problems of pesticide residues and environmental pollution caused by excessive application of pesticides cannot be ignored. Especially when powdery mildew breaks out in a concentrated manner, it is difficult for chemical agents to achieve a relatively ideal prevention and control effect, and repeated excessive use of agents will also produce drug resistance.

[0003] The changes in plant metabolites under disease stress are affected by both their genes and environmental factors, and are a direct reflection of the plant's physiological phenotype and biochemical levels. Disease-infected plants produce many metabolites that can improve plant disease resistance in a variety of metabolic processes. These metabolites are an important material basis for the plant immune system, and the remodeling of metabolic flux largely reflects the plant's response and defense to disease. Studies have found that different secondary metabolic pathways play an important role in disease defense in various ways in Arabidopsis, rice, corn, wheat and other plants. Therefore, screening wheat powdery mildew resistance metabolites, analyzing their biosynthesis and regulatory pathways, and disease resistance mechanisms are particularly important for the prevention and control of wheat powdery mildew. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the TaACT4-3 gene in regulating the content of plant phenolamine substances and / or plant powdery mildew resistance, so as to regulate the content of plant phenolamine substances and the powdery mildew resistance of plants without changing the quantitative traits of the plants.

[0005] The present invention provides the use of the TaACT4-3 gene in regulating the content of plant phenolamine substances and / or regulating the powdery mildew resistance of plants;

[0006] The amino acid sequence of the protein encoded by the TaACT4-3 gene is shown in SEQ ID NO:3.

[0007] Preferably, the regulating the content of plant phenolamine substances includes: overexpressing TaACT4-3 gene to increase the content of plant phenolamine substances.

[0008] Preferably, the phenolamine substances include one or more of coumaroyl agmatine, caffeoyl agmatine, feruloyl agmatine and sinapinic acid guabutine.

[0009] 4. The use according to claim 1, characterized in that the use also includes: not changing the quantitative traits of plants.

[0010] Preferably, the quantitative traits include one or more of plant height, tiller number, grain number and grain weight.

[0011] Preferably, the regulating the powdery mildew resistance of plants comprises: overexpressing the TaACT4-3 gene to improve the powdery mildew resistance of plants and prevent and control the powdery mildew of plants.

[0012] Preferably, the plant includes one or more of wheat, potato, rice and highland barley.

[0013] Preferably, the nucleotide sequence of the coding region of the TaACT4-3 gene is shown in SEQ ID NO:2.

[0014] Preferably, the full-length genomic nucleotide sequence of the TaACT4-3 gene is shown in SEQ ID NO: 1.

[0015] The present invention also provides a method for increasing the content of plant phenolamine substances and / or the powdery mildew resistance of plants, which comprises overexpressing the TaACT4-3 gene in plants; the amino acid sequence of the protein encoded by the TaACT4-3 gene is shown in SEQ ID NO:3.

[0016] Beneficial effects:

[0017] The present invention provides the use of the TaACT4-3 gene in regulating the content of plant phenolamine substances and / or regulating the resistance of plants to powdery mildew; the amino acid sequence of the protein encoded by the TaACT4-3 gene is shown in SEQ ID NO: 3. The present invention develops and utilizes the TaACT4-3 gene, and by overexpressing the TaACT4-3 gene, the lignin content and hydrogen peroxide content of the plant can be increased, the content of phenolamine substances in the plant, especially in wheat, can be effectively increased, and the resistance of the plant to powdery mildew is enhanced without affecting the original quantitative traits of the plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1This is the classification of metabolites induced by powdery mildew infection on leaves of wheat seedlings in natural populations (FC>3);

[0020] Figure 2 The result of genome-wide association analysis of phenolamine content in wheat is the location of TaACT4-3;

[0021] Figure 3 is the expression level of TaACT4-3 in different wheat varieties after being treated with pathogens;

[0022] Figure 4 Schematic diagram of the genetic transformation vector structure for overexpressing TaACT4-3 in wheat;

[0023] Figure 5 The results of the analysis of the tolerance of wild-type wheat and transgenic wheat overexpressing TaACT4-3 to powdery mildew;

[0024] Figure 6 The results of the analysis of phenolamine and lignin contents in wild-type wheat and transgenic wheat overexpressing TaACT4-3; ** indicates P<0.01, *** indicates P<0.001;

[0025] Figure 7 The results of the analysis of the contents of phenolamines and hydrogen peroxide in wild-type wheat and transgenic wheat overexpressing TaACT4-3; ** indicates P<0.01, *** indicates P<0.001;

[0026] Figure 8 The results of quantitative trait analysis of wild-type wheat and transgenic wheat overexpressing TaACT4-3; * indicates P<0.05, and ** indicates P<0.01. DETAILED DESCRIPTION

[0027] The present invention provides the use of TaACT4-3 gene in regulating the content of plant phenolamine substances and / or regulating the powdery mildew resistance of plants; the amino acid sequence of the protein encoded by the TaACT4-3 gene is shown in SEQ ID NO:3.

[0028] As an embodiment, the present invention regulates the content of plant phenolamines, including: overexpressing the TaACT4-3 gene to increase the content of plant phenolamines. As an embodiment, the present invention comprises one or more of coumaroyl agmatine, caffeoyl agmatine, feruloyl agmatine and sinapinic acid guabutinamine; as another embodiment, the present invention comprises coumaroyl agmatine, caffeoyl agmatine, feruloyl agmatine and sinapinic acid guabutinamine.

[0029] As an embodiment, the application of the present invention also includes: not changing the quantitative traits of the plant. As an embodiment, the quantitative traits of the present invention include one or more of plant height, tiller number, grain number and grain weight; as another embodiment, the quantitative traits of the present invention include plant height, tiller number, grain number and grain weight. As an embodiment, the tiller number of the present invention may be the effective tiller number. As an embodiment, the grain number of the present invention may be the number of grains per ear. As an embodiment, the grain weight of the present invention may be thousand-grain weight.

[0030] As an embodiment, the regulation of plant powdery mildew resistance of the present invention includes: overexpressing TaACT4-3 gene to improve plant powdery mildew resistance and prevent and control plant powdery mildew.

[0031] As one embodiment, the plant of the present invention includes one or more of wheat, potato, rice and barley; as another embodiment, the plant of the present invention is wheat.

[0032] As an embodiment, the coding region nucleotide sequence of the TaACT4-3 gene of the present invention is shown as SEQ ID NO: 2. As an embodiment, the full-length genomic nucleotide sequence of the TaACT4-3 gene of the present invention is shown as SEQ ID NO: 1.

[0033] The present invention provides a method for increasing the content of plant phenolamine substances and / or the powdery mildew resistance of plants, which comprises overexpressing the TaACT4-3 gene in plants; the amino acid sequence of the protein encoded by the TaACT4-3 gene is shown in SEQ ID NO:3.

[0034] As an embodiment, the plant of the present invention includes wheat. The present invention has no strict requirements on the method of overexpressing the TaACT4-3 gene, and conventional methods in the art can be used.

[0035] To further illustrate the present invention, the application of the TaACT4-3 gene provided by the present invention in regulating the content of plant phenolamine substances and / or plant powdery mildew resistance is described in detail below in conjunction with the drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] Acquisition of TaACT4-3 gene

[0038] 1. TaACT4-3 gene identification and structural analysis

[0039] The inventors found that after powdery mildew infected wheat seedlings in natural populations, the contents of phenyloxalic acid and phenylalanine in the downstream branch metabolic pathway of phenylalanine increased ( Figure 1), and the infected population coumaroylguatidine was localized to a gene cluster located on chromosome 2D of wheat ( Figure 2 ), and further quantitative PCR detection showed that the acyltransferase in this gene cluster was induced after 24 hours of pathogen treatment. Among them, the most obvious gene number induced in different wheat varieties (LW05: Chinese Spring (CS), JZ189: Avocet and JZ283: FARO) was TraesCS2D01G491100.1, named TaACT4-3 ( Figure 3 The full-length genome sequence of TaACT4-3 is 1597 bp (SEQ ID No: 1), the coding sequence is 1326 bp (SEQ ID No: 2), encoding 441 amino acids (SEQ ID No: 3), and the gene has no introns.

[0040]

[0041]

[0042] SEQ ID NO: 3: MKITVHSSKAVKPEYGACGVAPGCTADVVPLTVLDKA NFDTYISVIYAFHAPAPHNDVLEAGLARALVDYREWAGRLGVDANGDRAILLNDAGARFVEATADVALVSVMPLKPTSEVLSLHPSGDDGPEELMLIQVTRFPCGSIVVGFTTQHIVSDGRSTGNFFVAWSQATRGSAIDPVPVHDRASFFQPREPLHVEYEHRGVEFKPYKKVYDDIVCGDGDDEDDEVVVNKVHFSREFIS RLKAQASAGAPRPCSTLQCVVAHLWRRTMTMARGLDGGESTSVAIAVDGRARMSPQVPDGYTGNVILWARPTTTAGELVSRPVKHAVELISREVARINDGYFKSFIDFANSGAVEEERLVATADAAEMVLSPNIEVDSWLRIPFYDMDFGGGRPFFFMPSYLPVEGLLILLPSFLGDGSVDAYVPLFSRDMNTFKNCCYTLD*.

[0043] 2. Amplification of TaACT4-3 gene

[0044] Total RNA of Fielder in wheat varieties was extracted, reverse transcribed into cDNA, and amplified using specific primers (TaACT4-3-F: 5'-ATGAAGATCACCGTGCACTCGTCC-3', SEQ ID NO: 4; TaACT4-3-R: 5'-CTAGTCGAGGGTGTAGCAGCAGTT-3', SEQ ID NO: 5). The target fragment of PCR amplification was connected to the blunt-end vector, and the reaction product was transformed into Escherichia coli DH5α, and 1mL LB was added for recovery for 1h. 150μL of the recovered bacterial solution was applied to a plate containing AMP-resistant LA medium and cultured in a 37℃ incubator for 12h. Single clones were tested by PCR, and plasmids were extracted for sequencing. The PCR products were sequenced and analyzed to obtain the cDNA sequence of the TaACT4-3 gene. The 1326bp TaACT4-3 gene sequence was amplified from Fielder. After the sequence alignment was correct, homologous recombination was performed with the target vector respectively.

[0045] Example 2

[0046] Analysis of resistance of TaACT4-3 genetic material to wheat powdery mildew

[0047] 1. Construction of TaACT4-3 genetic transformation vector

[0048] The 1326 bp TaACT4-3 gene fragment amplified from Fielder in Example 1 was subjected to homologous recombination reaction with the target vector pLGY to obtain a recombinant vector. The vector map is shown in Figure 4 The vector is an Agrobacterium-mediated genetic transformation vector carrying a maize ubiquitin gene promoter with constitutive and overexpression characteristics.

[0049] 2. TaACT4-3 genetic transformation

[0050] Referring to the prior art (Hayta S, Smedley MA, Demir SU, et al. An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.) [J]. Plant Methods, 2019, 15: 1-15.), the plasmid correctly cloned in step 1 was introduced into the wheat variety Fielder through Agrobacterium EHA105 mediation and wheat genetic transformation system, and transgenic plants were obtained through pre-culture, infection, co-cultivation, screening of callus tissue with glufosinate resistance, differentiation, rooting, seedling hardening and transplanting.

[0051] 3. Analysis of the tolerance of TaACT4-3 genetic material to powdery mildew

[0052] (1) The DNA level of T0 transgenic wheat plants was tested to confirm the construction of TaACT4-3 positive plants. RNA samples and metabolic samples were taken from T3 positive transgenic wheat plants overexpressing TaACT4-3 and wild-type wheat plants that had grown to two leaves and one heart, and the expression level of TaACT4-3 and the content of phenolamine substances in the plants were analyzed. The results showed that compared with the control wild-type Fielder, the expression level of the TaACT4-3 gene in the positive transformed plants overexpressing TaACT4-3 was greatly increased ( Figure 5 A), the contents of phenolamine substances such as coumaroyl agmatine (Cou-agm), caffeoyl agmatine (caf-agm), feruloyl guabutinamine (fer-agm) and sinapinic guabutinamine (sin-agm) also increased significantly ( Figure 5 (B) shows that the constructed genetic material can be used for further research and analysis.

[0053] (2) Based on the conclusion of step (1), the mature spores of wheat powdery mildew were evenly shaken onto the leaves of T3 positive transgenic wheat plants overexpressing TaACT4-3 that had grown to two leaves and one heart, and the wheat powdery mildew was inoculated; 24 hours (hpi) and 96 hours (hpi) after infection, the leaves were stained with Coomassie Brilliant Blue and observed under a microscope. The results showed that compared with the wild type, the hyphae coverage area on the leaves of wheat overexpressing TaACT4-3 was reduced and the hyphae grew more slowly ( Figure 5 Middle E). Wheat plants overexpressing TaACT4-3 showed tolerance to powdery mildew, with reduced hyphae coverage on leaves and slower hyphae growth compared to the wild type ( Figure 5 C). Meanwhile, the ratio of powdery mildew to wheat DNA in wheat plants overexpressing TaACT4-3 was significantly lower than that in wild type ( Figure 5 Middle D). TaACT4-3 can improve wheat tolerance to powdery mildew.

[0054] Example 3

[0055] Analysis of the resistance mechanism of TaACT4-3 genetic material to wheat powdery mildew

[0056] 1. Analysis of lignin content in TaACT4-3 genetic material

[0057] With reference to the prior art (Fang H, Zhang F, Zhang C, et al. Function of hydroxycinnamoyltransferases for the biosynthesis of phenolamides in rice resistance to Magnaporthe oryzae [J]. Journal of Genetics and Genomics, 2022, 49 (8): 776-786.), RNA samples and metabolic samples were taken from the T3 generation positive wheat plants overexpressing TaACT4-3 and wild-type wheat plants grown to two leaves and one heart in Example 2, and the leaves were collected, the leaves were dried to constant weight, and precipitates were obtained after treatment with different buffers. The precipitates were dried in an oven to obtain protein-free cell wall components, the content of phenolamine substances was analyzed, and then the acetyl bromide method was used for determination, and the standard curve was generated using alkaline lignin ( Figure 6 B), the results are expressed as mg lignin / g cell wall. The results showed that compared with the wild-type control, the contents of phenolamine substances coumaroyl agmatine (Cou-agm), caffeoyl agmatine (caf-agm), feruloyl guatamine (fer-agm) and sinapinic guatamine (sin-agm) in the TaACT4-3 overexpressing positive transformed plants were significantly increased ( Figure 6A), the lignin content in wheat plants overexpressing TaACT4-3 was significantly higher than that in wild type ( Figure 6 Middle C).

[0058] 2. Analysis of hydrogen peroxide content in TaACT4-3 genetic material

[0059] The materials sampled in step 1 were treated with powdery mildew, and the mature spores of wheat powdery mildew were evenly shaken onto the wheat leaves. Samples were taken 24 hours after treatment, and the hydrogen peroxide in the leaves was measured using the Solebow kit (Cat. No.: BC3595). The results showed that compared with the wild-type control, the contents of phenolamine substances coumaroyl agmatine (Cou-agm), caffeoyl agmatine (caf-agm), feruloyl guatamine (fer-agm) and sinapinic guatamine (sin-agm) in the positive transformed plants overexpressing TaACT4-3 were significantly increased ( Figure 7 Middle A), the hydrogen peroxide content of wheat plants overexpressing TaACT4-3 after powdery mildew infection was significantly higher than that of the wild type, and the expression levels of genes related to hydrogen peroxide production were also significantly upregulated ( Figure 7 (middle B).

[0060] Example 4

[0061] Analysis of quantitative traits of TaACT4-3 genetic materials in the field

[0062] The T4 generation plants overexpressing TaACT4-3 and wild-type plants of Example 2 were planted in the field, and DNA sampling was performed at the seedling stage to confirm that all the families were positive. After the wheat matured, the plant height, number of effective tillers, number of grains per ear, and grain traits were statistically analyzed. The results showed that there was no significant difference in the quantitative traits between the TaACT4-3 overexpressing plants and the wild-type plants ( Figure 8 ).

[0063] Based on the above content, it can be seen that overexpression of the TaACT4-3 gene can effectively increase the content of phenolamines in plants (especially wheat) and enhance the resistance of plants to powdery mildew without changing the quantitative traits of the plants.

[0064] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of TaACT4-3 gene in regulating the content of plant phenolamines and / or regulating the resistance of plants to powdery mildew; The amino acid sequence of the protein encoded by the TaACT4-3 gene is shown in SEQ ID NO:

3.

2. The use according to claim 1, characterized in that: The regulating the content of plant phenolamine substances includes: overexpressing the TaACT4-3 gene to increase the content of plant phenolamine substances.

3. The use according to claim 2, characterized in that: The phenolamine substances include one or more of coumaroyl agmatine, caffeoyl agmatine, feruloyl agmatine and sinapinic acid guabutine.

4. The use according to claim 1, characterized in that: The application also includes: not changing the quantitative traits of plants.

5. The use according to claim 4, characterized in that: The quantitative traits include one or more of plant height, tiller number, grain number and grain weight.

6. The use according to claim 1, characterized in that: The regulating plant powdery mildew resistance comprises: overexpressing the TaACT4-3 gene to improve the plant powdery mildew resistance and prevent and control the plant powdery mildew.

7. The use according to claim 1, characterized in that: The plants include one or more of wheat, potato, rice and highland barley.

8. The use according to any one of claims 1 to 7, characterized in that: The nucleotide sequence of the coding region of the TaACT4-3 gene is shown in SEQ ID NO:

2.

9. The use according to claim 8, characterized in that: The full-length genomic nucleotide sequence of the TaACT4-3 gene is shown in SEQ ID NO:

1.

10. A method for increasing the content of plant phenolamines and / or the resistance of plants to powdery mildew, characterized in that: The TaACT4-3 gene is overexpressed in plants; the amino acid sequence of the protein encoded by the TaACT4-3 gene is shown in SEQ ID NO:3.

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