阅读说明：本技术 小麦病程相关蛋白TaPR1a基因及其在小麦抗条锈病、叶锈病中的应用 (Wheat disease course related protein TaPR1a gene and application thereof in wheat stripe rust and leaf rust resistance ) 是由 王逍冬 赵姣洁 毕伟帅 赵淑清 苏君 庞书勇 于秀梅 刘大群 于 2020-07-02 设计创作，主要内容包括：本发明提供了小麦病程相关蛋白TaPR1a基因及其在小麦抗条锈病、叶锈病中的应用。本发明涉及基因序列,具体公开了小麦病程相关蛋白TaPR1a基因,其核苷酸序列如SEQ ID No.1所示,以及过表达TaPR1a基因的小麦转基因材料的制备过程。本发明通过实验验证TaPR1a基因可显著提高小麦对小麦条锈病和小麦叶锈病的抗性水平。(The invention provides a wheat disease course related protein TaPR1a gene and application thereof in wheat stripe rust and leaf rust resistance. The invention relates to a gene sequence, and particularly discloses a wheat disease course related protein TaPR1a gene, a nucleotide sequence of which is shown in SEQ ID No.1, and a preparation process of a wheat transgenic material for over-expressing TaPR1a gene. Experiments prove that the TaPR1a gene can obviously improve the resistance level of wheat to wheat stripe rust and wheat leaf rust.)

1. The wheat disease course related protein TaPR1a gene is characterized in that the nucleotide sequence is shown in SEQ ID No. 1.

2. The method for cloning the gene sequence as claimed in claim 1, wherein the gene sequence is cloned by PCR method using primers and using cDNA of wheat JW1 as a template.

3. The nucleotide sequence of the primer is as follows:

TaPR1a-ORF-F:GGTACCATGGAGACGCCCAAGCTGGC

TaPR1a-ORF-R:ACTAGTTTAGTATGGTTTCTGTCCAATGACATTC。

4. an expression vector comprising the gene sequence of claim 1.

5. The expression vector of claim 4, wherein the expression vector is a eukaryotic expression vector.

6. The expression vector of claim 5, wherein the expression vector is the wheat transgene vector pLGY-02.

7. A host comprising the expression vector of any one of claims 4 to 6.

8. Use of the gene sequence of claim 1 for modulating disease resistance of a plant to wheat stripe rust and wheat leaf rust.

9. Use according to claim 8, wherein the plant is Triticum aestivum.

Technical Field

The invention belongs to the technical field of biological gene engineering, and relates to a wheat disease course related protein TaPR1a gene and application thereof in wheat stripe rust and leaf rust resistance.

Background

The quality and the yield of common wheat as a main grain crop seriously influence the grain safety and the social stability of China, and the high yield and the stable yield of wheat have important significance for the agricultural development of China. Wheat stripe rust and wheat leaf rust caused by Puccinia striiformis f.sp.tritici and Puccinia triticina (Puccinia triticina) are important fungal diseases which seriously affect wheat production in China. In recent years, due to the increase of planting density and the change of agricultural cultivation system, the occurrence of wheat stripe rust and leaf rust becomes more and more serious, and the quality and safety of food in China are seriously influenced. The pathogenic bacteria of the wheat stripe rust and the leaf rust have the characteristic of high mutation frequency, and physiological races can complete multiple mutations in a short time, so that a wheat variety with single resistance easily loses resistance in a short time. Therefore, the development of new disease-resistant germplasm resources is necessary.

The disease course related Protein (PR) is a general name of a class of proteins induced and accumulated by plants after coping with biotic and abiotic stresses, is widely existed, and has extremely strong conservation in sequence characteristics and protein functions. PR proteins are an important component of the defense system of plants. PR genes were originally found primarily because they are expressed in large amounts when plants are infected with pathogens, often acting downstream of the plant's disease response, and in many cases directly limiting pathogen infection. However, recent studies have shown that PR genes play a role in plant senescence, injury, abiotic stress, hormonal treatment, and even normal growth and development.

The plant PR genes reported at present belong to 18 gene families. Among them, the PR1 gene is considered as an indicator gene for activation of a plant's defense response against various pathogens. The PR1 gene encodes a small molecule secretory protein with CAPE1 short peptide at the C end, and can be used as a damage-inducing molecule pattern (DAMP) to induce plants to generate a basic disease resistance response (PTI). PR1 proteins have been reported to be involved in a wide variety of biological processes, including sterol binding, defense signaling, and targeted inhibition of plant pathogen effector proteins. For example, the wheat TaPR1 protein was found to interact directly with the ToxA toxin of the necrophoretic pathogen and mediate a ToxA-induced necrosis reaction in susceptible wheat strains.

Disclosure of Invention

The invention aims to provide a wheat disease course related protein TaPR1a gene and application thereof in wheat stripe rust resistance and leaf rust resistance.

The invention provides a wheat disease course related protein TaPR1a gene, the nucleotide sequence of which is shown in SEQ ID No.1, or the nucleotide sequence which is formed by substituting, deleting and/or adding one or more nucleotides and is derived from SEQ ID No.1 and codes an amino acid sequence with the same function.

The invention provides a cloning method of the gene sequence, which comprises the following steps: using cDNA of common wheat spring wheat material 'JW 1' as a template, and cloning by using a polymerase chain reaction PCR method by using primers to obtain the gene sequence;

the invention also provides an expression vector containing the gene sequence. Preferably, the expression vector is a eukaryotic expression vector, and more preferably is a pLGY-02(Ubi:: Gene, T-DNA) vector. For example, the eukaryotic expression vector may be obtained by cloning the aforementioned gene sequence into a pLGY-02 vector.

The present invention provides a host containing the aforementioned expression vector. Alternatively, the host may be escherichia coli, agrobacterium, wheat, or the like. For example, the wheat may be the common wheat spring wheat variety "JW 1".

The invention provides application of the gene sequence in regulating and controlling the disease resistance of plants to wheat stripe rust and wheat leaf rust. Preferably, the plant is wheat. More preferably, the common wheat is common wheat spring wheat variety "JW 1".

The invention has the beneficial effects that: the invention provides a wheat disease course related protein TaPR1a gene sequence and a preparation process of a wheat transgenic material for over-expressing a TaPR1a gene. Experiments prove that the disease resistance of the wheat transgenic material to the wheat stripe rust and the leaf rust is obviously improved.

Drawings

FIG. 1 shows that the disease resistance level of wheat stripe rust CYR32 inoculated with the wheat transgenic material over-expressing TaPR1a gene is obviously improved.

FIG. 2 shows that the disease resistance level of Puccinia triticina THTT inoculated with the wheat transgenic material over-expressing TaPR1a gene is obviously improved.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Plant material: common wheat spring wheat variety "JW 1".

Strain and carrier: coli TOP10 competent cells (CB104) were purchased from Tiangen Biochemical technology, Inc. (Beijing). The T cloning vector pGEM-Teasy was purchased from Beijing Quanyujin Biotechnology, Inc. Agrobacterium GV3101 and wheat transgenic vector pLGY-02 were maintained by the present laboratory. The puccinia striiformis toxicity physiological race CYR32 and the puccinia striiformis toxicity physiological race THTT are preserved by North river agricultural university.

The main reagents are as follows: agarose was purchased from SIGMA; 2 XPremix Taq enzyme was purchased from Shijieki Biotechnology Co., Ltd, restriction enzymes Kpn I, Spe I (TaKaRa engineering Co., Ltd., Boehringer Mannheim); sucrose, glucose, tryptone, agar powder, Tween-20, isopropanol, glycerol, beta-mercaptoethanol, sodium chloride, sodium hydroxide, absolute ethanol, boric acid, Tris-HCl and other reagents are purchased from Ministry of Wanke chemical reagents. The AL2000 DNA Marker, the plasmid small-scale extraction kit, the gel recovery and purification kit are purchased from Biotechnology, Inc., the QIAGEN plant total RNA extraction kit is purchased from Tiangen Biotechnology, Inc., and the SYBR Premix Dimer Eraser fluorescence quantitative kit and the reverse transcription kit are purchased from Beijing all-purpose gold biotechnology, Inc.

The main apparatus is as follows: applied Biosystems Veriti Thermal Cycler PCR amplification instrument (ThermoFisher), WH-861 vortex mixer (science and education instruments, taicang), high speed refrigerated Centrifuge 5810R (Eppendorf corporation), small high speed desktop Centrifuge 5415D (Eppendorf corporation), ultra clean bench (AIR TECH corporation), constant temperature shaking incubator (shanghai su kushui ltd.), SX-500 sterilization pot (TOMY corporation), ice maker (SCOTSMAN corporation), molar element type ultrapure water machine (shanghai morganic instruments ltd.), microwave oven (Galanz corporation), water bath pot (beijing maxuanchi instruments), micro amount (Eppendorf corporation), SONY CarlZeiss varia Sonnar camera (SONY), LightCycler96 real time fluorescence quantitative PCR instrument (Roche corporation), sample grinder, and the like.