Application of TaF5H3 protein or coding gene thereof in regulation and control of wheat lignin precursor substance content

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to application of TaF5H3 protein or a coding gene thereof in regulating and controlling the content of wheat lignin precursor substances. The invention provides an application of a TaF5H3 protein or an encoding gene thereof in regulating and controlling the content of a wheat lignin precursor substance. The lignin precursor substance is prepared from one or more of 5-hydroxy ferulic acid, 5-hydroxy coniferyl aldehyde and 5-hydroxy coniferyl alcohol; the amino acid sequence of the TaF5H3 protein is as shown in SEQ ID NO: 3. The TaF5H3 protein or the coding gene of the TaF5H3 protein is overexpressed in wheat, so that the content of wheat lignin precursors 5-hydroxyferulic acid, 5-hydroxyconiferyl aldehyde and 5-hydroxyconiferyl alcohol can be increased, and the TaF5H3 protein or the coding gene of the TaF5H3 protein possibly has a certain effect on wheat disease resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of TaF5H3 protein or its encoding gene in regulating the content of wheat lignin precursor substances. Background Art

[0002] Wheat powdery mildew, caused by Blumeria graminis f.sp.Tritici (Bgt), is a global fungal disease. Infection of wheat by powdery mildew causes leaf dieback and reduces tiller number, ear set rate, number of grains per ear, and 1000-grain weight, seriously threatening wheat yield and quality. Currently, the prevention and control of wheat powdery mildew in production still mainly relies on spraying with chemical agents and reasonable dense planting. However, the use of chemical agents such as triadimenol and triadimefon not only increases field input costs, but also causes pesticide residues and environmental pollution caused by excessive application of pesticides, which cannot be ignored. Especially when powdery mildew breaks out in a concentrated manner, chemical agents are difficult to achieve a relatively ideal prevention and control effect, and repeated excessive use of agents will also produce drug resistance.

[0003] Different secondary metabolic pathways play a crucial role in disease defense in various ways. While the cuticle in the plant epidermis and lignin and callose in the cell walls do not inherently possess antimicrobial activity, their mechanical strength and tolerance to pathogens allow them to participate in constitutive and induced resistance in plants, preventing pathogens from invading and further spreading. Lignin is a complex phenolic polymer that is indegradable by most microorganisms, thus acting as a physical barrier in plants' defense against pathogen infection.

[0004] The synthesis of lignin is a complex process. Its synthesis pathway primarily uses phenylalanine as a substrate, undergoing a series of biochemical reactions to form different types of lignin monomers, which then polymerize to form the final lignin polymer. Currently, several important genes involved in lignin monomer synthesis have been cloned, and with the help of genetic engineering and other research techniques, the functions of these genes in plant lignin metabolism have been verified. Although lignin anabolism has been studied in depth, its research has primarily focused on plants such as Arabidopsis, tobacco, alfalfa, and corn, with relatively few reports on the regulation of wheat lignin anabolism. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of TaF5H3 protein or its encoding gene in regulating the content of wheat lignin precursors, regulating the content of one or more of the wheat lignin precursors 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol, thereby increasing the lignin content and improving powdery mildew resistance.

[0006] The present invention provides the use of TaF5H3 protein or its encoding gene in regulating the content of wheat lignin precursor substances;

[0007] The lignin precursor comprises one or more of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol;

[0008] The amino acid sequence of the TaF5H3 protein is shown in SEQ ID NO: 3;

[0009] Preferably, the regulating the content of wheat lignin precursor substances includes: overexpressing TaF5H3 protein or its encoding gene to increase the content of wheat lignin precursor substances.

[0010] Preferably, the method of increasing the content of lignin precursors in wheat is to increase the content of lignin precursors in wheat leaves.

[0011] Preferably, the nucleotide sequence of the coding region of the gene encoding the TaF5H3 protein is shown in SEQ ID NO: 2.

[0012] Preferably, the full-length genomic nucleotide sequence of the encoding gene is shown as SEQ ID NO: 1.

[0013] The present invention also provides the use of a recombinant strain comprising the TaF5H3 gene in increasing the content of lignin precursors in wheat;

[0014] The lignin precursor comprises one or more of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol;

[0015] The recombinant strain includes the TaF5H3 gene and an initial strain; the initial strain includes Agrobacterium; the amino acid sequence of the TaF5H3 protein encoded by the TaF5H3 gene is shown in SEQ ID NO: 3.

[0016] The present invention also provides the use of TaF5H3 protein or its encoding gene or a recombinant strain containing the TaF5H3 protein encoding gene in adding a hydroxyl group to the 5th position of the benzene ring of ferulic acid to generate 5-hydroxyferulic acid;

[0017] The amino acid sequence of the TaF5H3 protein is shown in SEQ ID NO: 3.

[0018] Preferably, the recombinant strain includes the gene encoding the TaF5H3 protein and an initial strain; the initial strain includes yeast.

[0019] The present invention also provides a method for increasing the content of lignin precursors in wheat, comprising the following steps: overexpressing the TaF5H3 gene in wheat; the amino acid sequence of the TaF5H3 protein encoded by the TaF5H3 gene is shown in SEQ ID NO: 3;

[0020] The lignin precursor includes one or more of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol.

[0021] Beneficial effects:

[0022] The present invention provides the use of a TaF5H3 protein or a gene encoding it for regulating the content of lignin precursors in wheat; the lignin precursors include one or more of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde, and 5-hydroxyconiferyl alcohol; the amino acid sequence of the TaF5H3 protein is shown in SEQ ID NO: 3. The present invention demonstrates that TaF5H3 can add a hydroxyl group to the 5th position of the benzene ring of ferulic acid, coniferyl aldehyde, and coniferyl alcohol to produce 5-hydroxyferulic acid, 5-hydroxyconiferyl aldehyde, and 5-hydroxyconiferyl alcohol. Overexpression of the TaF5H3 gene in wheat can increase the content of the wheat lignin precursors 5-hydroxyferulic acid, 5-hydroxyconiferyl aldehyde, and 5-hydroxyconiferyl alcohol, and has a potential role in wheat resistance to powdery mildew. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0024] Figure 1 This is a classification diagram of metabolites induced by powdery mildew infection in wheat seedling leaves in natural populations (FC>3);

[0025] Figure 2 The results of the genome-wide association analysis of phenolamine content in wheat are shown in Figure 3.

[0026] Figure 3 represents the expression levels of TaF5H3 in different wheat lines after being infected with powdery mildew; LW05 is Chinese Spring (CS) wheat, JZ189 is Avocet wheat, and JZ283 is FARO wheat;

[0027] Figure 4 This is the chromatogram of the reaction results of the recombinant protein TaF5H3-pYES2 in yeast;

[0028] Figure 5 Schematic diagram of the genetic transformation vector structure for overexpressing TaF5H3 in wheat;

[0029] Figure 6The expression levels of TaF5H3 gene in wild-type wheat and transgenic wheat overexpressing TaF5H3, as well as the contents of wheat lignin precursors 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde, and 5-hydroxyconiferyl alcohol; * indicates P < 0.05, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0030] The present invention provides the use of a TaF5H3 protein or a gene encoding the same in regulating the content of lignin precursors in wheat; the lignin precursors include one or more of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde, and 5-hydroxyconiferyl alcohol; the amino acid sequence of the TaF5H3 protein is shown in SEQ ID NO: 3;

[0031] In one embodiment, the present invention regulates the content of wheat lignin precursors by overexpressing the TaF5H3 protein or its encoding gene to increase the content of wheat lignin precursors. In one embodiment, the present invention increases the content of wheat lignin precursors by increasing the content of wheat leaf lignin precursors.

[0032] In one embodiment, the nucleotide sequence of the coding region of the gene encoding the TaF5H3 protein of the present invention is shown in SEQ ID NO: 2. In one embodiment, the full-length genomic nucleotide sequence of the gene encoding the TaF5H3 protein of the present invention is shown in SEQ ID NO: 1.

[0033] The present invention also provides the use of a recombinant strain comprising the TaF5H3 gene for increasing the content of lignin precursors in wheat; the lignin precursors comprise one or more of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde, and 5-hydroxyconiferyl alcohol; the recombinant strain comprises the TaF5H3 gene and an initial strain; the initial strain comprises Agrobacterium; the amino acid sequence of the TaF5H3 protein encoded by the TaF5H3 gene is shown in SEQ ID NO: 3. As one embodiment, the recombinant strain of the present invention further comprises an initial vector, into which the TaF5H3 gene is inserted to form a recombinant vector. As one embodiment, the initial vector of the present invention comprises pLGY.

[0034] The present invention also provides the use of TaF5H3 protein or its encoding gene or a recombinant strain containing the TaF5H3 protein encoding gene in adding a hydroxyl group to the 5th position of the benzene ring of ferulic acid to generate 5-hydroxyferulic acid; the amino acid sequence of the TaF5H3 protein is shown in SEQ ID NO: 3.

[0035] In one embodiment, the recombinant strain of the present invention comprises a gene encoding the TaF5H3 protein and an initial strain; the initial strain comprises yeast. In one embodiment, the recombinant strain of the present invention also comprises an initial vector, into which the TaF5H3 gene is inserted to form a recombinant vector. In one embodiment, the initial vector of the present invention comprises pYES2.

[0036] The present invention also provides a method for increasing the content of lignin precursors in wheat, comprising the following steps: overexpressing the TaF5H3 gene in wheat; the amino acid sequence of the TaF5H3 protein encoded by the TaF5H3 gene is shown in SEQ ID NO: 3; and the lignin precursors include one or more of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol.

[0037] The present invention has no strict requirements on the method of overexpressing the TaF5H3 gene, and conventional methods in the art can be used.

[0038] To further illustrate the present invention, the application of the TaF5H3 protein or its encoding gene provided by the present invention in regulating the content of wheat lignin precursor substances is described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Acquisition of TaF5H3 gene

[0041] 1. TaF5H3 gene identification and structural analysis

[0042] The inventors found that after powdery mildew infects wheat seedlings in natural populations, the contents of phenyloxalic acid and phenylalanine in the downstream metabolic pathway of phenylalanine increase ( Figure 1 ), coumaroylguatinamide in the infected population was located in a gene cluster located on chromosome 2D of wheat ( Figure 2 ), further quantitative PCR detection revealed that the gene numbered TraesCS2D01G490500 in this gene cluster was induced in different wheat varieties after pathogen treatment ( Figure 3 The TraesCS2D01G490500 gene was named TaF5H3. The full-length genomic sequence is 2447 bp (SEQ ID NO: 1), the coding sequence is 1602 bp (SEQ ID NO: 2), encoding 533 amino acids (SEQ ID NO: 3). The gene has one intron and two exons. The specific sequence information is as follows:

[0043]

[0044]

[0045]

[0046] 2. Amplification of the TaF5H3 gene

[0047] (1) Total RNA was extracted from Fielder wheat and reverse transcribed into cDNA. PCR amplification was performed using Fly fast high-fidelity DNA polymerase and specific primers (TaF5H3-F: 5'-ATGGTGGGCTTGGCCAAGAT-3', SEQ ID NO: 4; TaF5H3-R: 5'-TCACACCTTGTGCGTGGCAC-3', SEQ ID NO: 5) according to the instructions to obtain PCR amplification products.

[0048] (2) The PCR amplification product obtained in step (1) was connected to the blunt-end vector to obtain a ligation product; wherein, the ligation system was 1.75 μL of PCR product, 0.25 μL of 10× Enhancer, and 0.5 μL of pTOPO-T simple vector; the ligation conditions were 25°C for 10 min.

[0049] (3) The ligation product obtained in step (2) was transformed into Escherichia coli DH5α and revived in 1 mL of LB for 1 hour. 150 μL of the revived bacterial solution was plated on a plate containing LA medium containing AMP resistance and incubated in a 37°C incubator for 12 hours. A single clone was selected for PCR detection, and the plasmid was extracted and sequenced to obtain the cDNA sequence of the TaF5H3 gene. Sequencing analysis of the PCR product showed that a 1602 bp TaF5H3 gene sequence was amplified from Fielder and the sequence alignment was correct.

[0050] Example 2

[0051] (1) Referring to the prior art (Selection of a subspecies-specific diterpene gene cluster implicated in rice disease resistance), the 1602 bp TaF5H3 gene fragment amplified from Fielder in Example 1 was constructed into the pYES2 expression vector by homologous recombination. The constructed vector was transformed into the yeast strain WAT11 using the lithium acetate method, and the recombinant cells were cultured at 30°C in SC basal medium containing 2% glucose. For protein induction, the yeast cells were collected and resuspended in SD / -Ura medium containing 2% galactose instead of glucose and cultured at 30°C for 2 days. The cells were collected by centrifugation, resuspended in 50 mM Tris-HCl (pH 7.5, 1 mM EDTA, 600 mM sorbitol) buffer, and subjected to high-pressure disruption. After disruption, the cells were centrifuged at 12,000 g for 60 min under low temperature conditions. The resulting supernatant was centrifuged at 120,000 g for 90 min, and the precipitate was collected to obtain the protein expressed by the recombinant strain. A blank vector was used as a control group to obtain the protein expressed by the empty vector strain.

[0052] (2) The protein expressed by the recombinant strain obtained in step (1) and the protein expressed by the empty strain were used to perform an in vitro enzymatic activity reaction of ferulic acid 5-hydroxylase. The reaction system was 500 μL: 1 mg of the protein to be tested, 500 mM NADPH, 200 μM ferulic acid (substrate) and the balance 100 mM Tris-HCl (pH 7.5, 1 mM EDTA, 20% glycerol). The reaction conditions were: 30° C., 120 rpm, and then 500 μL of ethyl acetate was added to terminate the reaction and vortexed. The supernatant was centrifuged and analyzed by LC-MS using ferulic acid (substrate) and pentahydroxyferulic acid (product) as standards. The results are as follows: Figure 4 As shown. Among them, Figure 4 From top to bottom are the test results of the standard, recombinant strain expressed protein and empty strain expressed protein. Figure 4 It can be seen that the recombinant protein TaF5H3 expressed in yeast can add a hydroxyl group to the 5th position of the benzene ring of ferulic acid to generate the product 5-hydroxyferulic acid.

[0053] Example 3

[0054] Regulation of wheat lignin precursors 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol by TaF5H3 genetic material

[0055] 1. Construction of TaF5H3 genetic transformation vector

[0056] The 1602 bp TaF5H3 gene fragment amplified from Fielder in Example 1 was subjected to homologous recombination reaction with the target vector pLGY to obtain the recombinant vector TaF5H3-pLGY. The mass spectrum is shown in FIG. Figure 5 The recombinant vector TaF5H3-pLGY is an Agrobacterium-mediated genetic transformation vector carrying a maize ubiquitin gene promoter with constitutive and overexpression characteristics.

[0057] 2. 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-mediated and wheat genetic transformation system. After pre-culture, infection, co-cultivation, and screening of callus tissue with glufosinate resistance, differentiation, rooting, seedling hardening, and transplanting, TaF5H3 overexpressing transgenic plants were obtained.

[0058] 3. Analysis of gene expression levels in TaF5H3 overexpressing transgenic materials and the contents of wheat lignin precursors 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde, and 5-hydroxyconiferyl alcohol

[0059] The DNA level of the T0 generation transgenic wheat plants obtained in step 2 was tested to confirm the construction of TaF5H3 positive plants. Leaf RNA samples and metabolic samples were taken from the T3 generation positive transgenic wheat plants overexpressing TaF5H3 and wild-type wheat plants that had grown to two leaves and one heart, and the expression level of TaF5H3 in the plants, as well as the content of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol were analyzed. The results showed that compared with the control wild-type Fielder, the expression level of the TaF5H3 gene in the TaF5H3 overexpressing positive transformed plants was significantly increased ( Figure 6 A), the contents of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol increased significantly ( Figure 6 Middle B).

[0060] Based on the above content, it can be seen that the TaF5H3 provided by the present invention can increase the content of lignin monomer precursors 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol in wheat.

[0061] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. Application of TaF5H3 protein or its encoding gene in regulating the content of wheat lignin precursors; The lignin precursor comprises one or more of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol; The amino acid sequence of the TaF5H3 protein is shown in SEQ ID NO:

3.

2. The use according to claim 1, characterized in that The method for regulating the content of wheat lignin precursor substances includes overexpressing TaF5H3 protein or its encoding gene to increase the content of wheat lignin precursor substances.

3. The use according to claim 2, characterized in that The method of increasing the content of lignin precursor substances in wheat is to increase the content of lignin precursor substances in wheat leaves.

4. The use according to any one of claims 1 to 3, characterized in that The nucleotide sequence of the coding region of the gene encoding the TaF5H3 protein is shown in SEQ ID NO:

2.

5. The use according to claim 5, characterized in that The full-length genomic nucleotide sequence of the coding gene is shown in SEQ ID NO:

1.

6. Use of a recombinant strain containing the TaF5H3 gene to increase the content of lignin precursors in wheat; The lignin precursor comprises one or more of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol; The recombinant strain includes the TaF5H3 gene and an initial strain; the initial strain includes Agrobacterium; the amino acid sequence of the TaF5H3 protein encoded by the TaF5H3 gene is shown in SEQ ID NO:

3.

7. Use of TaF5H3 protein or its encoding gene or a recombinant strain containing the TaF5H3 protein encoding gene in the addition of a hydroxyl group to the 5-position of the benzene ring of ferulic acid to produce 5-hydroxyferulic acid; The amino acid sequence of the TaF5H3 protein is shown in SEQ ID NO:

3.

8. The use according to claim 8, characterized in that The recombinant strain includes the coding gene of the TaF5H3 protein and an initial strain; the initial strain includes yeast.

9. A method for increasing the content of lignin precursors in wheat, characterized in that: The method comprises the following steps: overexpressing the TaF5H3 gene in wheat; the amino acid sequence of the TaF5H3 protein encoded by the TaF5H3 gene is shown in SEQ ID NO: 3; The lignin precursor includes one or more of 5-hydroxyferulic acid, 5-hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol.

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