CN111662913B

CN111662913B - Wheat disease course related protein TaPR1a gene and application thereof in wheat stripe rust and leaf rust resistance

Info

Publication number: CN111662913B
Application number: CN202010629140.0A
Authority: CN
Inventors: 王逍冬; 赵姣洁; 毕伟帅; 赵淑清; 苏君; 庞书勇; 于秀梅; 刘大群
Original assignee: Heibei Agricultural University
Current assignee: Heibei Agricultural University
Priority date: 2020-07-02
Filing date: 2020-07-02
Publication date: 2022-06-24
Anticipated expiration: 2040-07-02
Also published as: CN111662913A

Abstract

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.

Description

Wheat disease course related protein TaPR1a gene and application thereof in wheat stripe rust and leaf rust resistance

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, it is necessary to discover new disease-resistant germplasm resources.

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 process 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".

Bacterial 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 (Thermo Fisher), WH-861 vortex mixer (science and education instruments, taicang), high-speed refrigerated Centrifuge 5810R (Eppendorf corporation), small-sized high-speed desktop Centrifuge 5415D (Eppendorf corporation), ultra-clean bench (AIR TECH corporation), constant temperature shaking incubator (shanghai su kusho ltd.), SX-500 sterilization pot (TOMY corporation), ice maker (SCOTSMAN corporation), molar element type ultrapure water machine (shanghai mor scientific instruments ltd.), microwave oven (Galanz corporation), water bath pot (beijing majo instruments), micro amount (Eppendorf corporation), SONY Carl Zeiss varia Sonnar camera (SONY), LightCycler96 real-time fluorescence quantitative PCR instrument (Roche corporation), sample grinder, and the like.

Example 1 cloning of wheat disease progression-associated protein TaPR1a Gene

Extracting RNA of wheat leaves: RNA Extraction was performed using the RNA Extraction Kit (QIAGEN, Hilden, Germany). A second leaf blade sample of spring wheat material JW1 in seedling stage is rapidly ground into powder in a sterilized mortar by liquid nitrogen for later use. Preparing Buffer RLT mixed liquor, adding 10 mu L of beta-mercaptoethanol into each ml of Buffer RLT, mixing the mixture for use at present, and placing the mixture on ice after mixing. The ground RNA sample was taken out of the liquid nitrogen, and 500. mu.L of Buffer RLT mixture was added quickly, shaken well, and centrifuged at 10000g for 2 min. The supernatant was pipetted with a pipette and transferred to a purple spin column and centrifuged at 10000g for 1 min. Transferring the collected liquid into a pink centrifugal column, adding pre-cooled absolute ethyl alcohol (the addition amount is 1/2 of the collected liquid), reversing, uniformly mixing, and standing to separate out nucleic acid. The mixture was instantaneously separated for 30 seconds, and the collected liquid was decanted. Add 700. mu.L of RW1 (washed protein) and snap-off for 30s and discard the pool. 500 μ L of Buffer RPE (44 mL absolute ethanol was added before use) was added and the mixture was centrifuged off instantaneously for 30s and the collected solution was decanted off. Repeating the above steps once, centrifuging at 10000g for 2 min. The column was replaced with a new 2mL collection tube and left to empty for 1 min. After the centrifugation, the pink centrifugal column was put into a 1.5mL centrifuge tube provided in the kit itself, and 30. mu.L of RNase-free water was added to the center of the adsorption membrane using a pipette, and the mixture was centrifuged for 1 min. The collected RNA samples were stored and the RNA concentration was measured by Nanodrop ultramicro spectrophotometry.

Reverse transcription of wheat cDNA: for extracting the obtained RNA sample

First-Strand cDNA Synthesis Supermix reverse transcription kit (all gold) was inverted to generate cDNA. All RNA samples were normalized to 1000ng using RNase free ddH₂Make up to 8. mu.L of O, and add 1. mu.L of Oligo (dT)12-18Primer (50. mu.L)M), blowing, sucking, uniformly mixing, and placing into a PCR instrument at 65 ℃/5min and 4 ℃/2 min. Then 10. mu.L of 2 × ES Reaction Mix, 1. mu.L was added

Mixing the RT/RI Enzyme Mix, putting the mixture into a PCR instrument again at 42 ℃/15min, and heating the mixture for 5s at 85 ℃ to ensure that

RT/RI lost activity and was stored at 4 ℃. The resulting cDNA template was purified with sterile water at a rate of 1: 5, and storing at-20 ℃ for later use.

And (3) PCR amplification: wheat cDNA is taken as a template, and PCR amplification is carried out by using a TaPR1a gene amplification primer. The primer information is shown in SEQ ID No. 2-3. PCR amplification System: cDNA: mu.L of 1. mu.L of each F/R primer, 0.5. mu.L of 2 XPromix Taq 12.5. mu.L of ddH₂And O is supplemented to 25 mu L. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing: 60 ℃/30s, extension: 72 deg.C/1 min, 35 cycles, and finally an extension of 7min at 72 deg.C.

Electrophoresis of PCR products and recovery and purification of target fragments: preparing 1% agarose gel and 0.5 × TBE electrophoresis solution, wherein the electrophoresis conditions are as follows: U110V, I mA 100mA, P90W, Time min 30 min. The target fragment was cut into gel under a gel cutter and recovered with a gel recovery kit (Bio-chemical Co.).

Construction of cloning vector: connecting the recovered product to a pGEM-Teasy vector, wherein the reaction system is as follows: mu.L of 2 XBuffer Buffer, 3.0. mu.L of PCR gel recovery product, 1.0. mu. L T4DNA ligase, and 1.0. mu.L of pGEM T-easy vector, and centrifuging to mix the reagents well, and ligating at 22 ℃ for at least 1h or 4 ℃ overnight.

Transformation of the recombinant plasmid: thawing DH5 alpha competent cell on ice for 5 min; adding all the connecting liquid, mixing, and ice-cooling for 20 min; performing heat shock on the metal bath for 60 s; adding 150 mu L of LB liquid culture medium after ice bath for 5 min; shaking at 37 deg.C and 200rpm for 50 min; and (3) coating a plate (Amp resistant solid culture medium) on an ultra-clean workbench, airing, sealing by using a sealing film, and culturing overnight in an incubator at 37 ℃.

Screening of recombinant plasmids: 8 spots (half of each spot is reserved) are picked, positive and negative controls are made, and a universal primer T7-F/SP6-R or a cross primer is used for PCR identification; the positive colonies were selected for plaque shaking, inoculated into 6mL of Amp resistant liquid LB medium in a 10mL sterilized centrifuge tube, and shake cultured overnight at 200rpm in a shaker at 37 ℃.

And (3) plasmid extraction:

the DNA plasmid miniprep kit from Sangon Biotech company extracts plasmids. The cultured cell suspension was aspirated into a 2mL centrifuge tube (500. mu.L), and then 500. mu.L of 50% glycerol was added thereto, followed by storing the glycerol cells at-20 ℃. The remaining bacteria solution is centrifuged at 8000g for 2min to collect the bacteria, and the supernatant is decanted. 250. mu.L of Buffer P1 (RNase A was added before use and stored at 4 ℃) was added thereto, and the cells were suspended thoroughly by shaking. Add 250. mu.L Buffer P2 (color developing reagent added before use, stored at 28 ℃), mix by inversion, and let stand for 2 min. mu.L of Buffer P3 was added using a pipette and turned upside down to completely disappear the blue color until white floc appeared. Centrifuging at 12000g for 8min, precipitating impurities into the bottom of the tube, transferring the supernatant into an adsorption column, instantaneously separating, and discarding the waste liquid. Then 500. mu.L of Wash solution was added to the adsorption column, followed by flash separation and waste solution discard. The Wash solution step is repeated once. The column was centrifuged for 1 min. The column was transferred to a sterile 1.5mL centrifuge tube, 40. mu.L of an Elution buffer preheated at 60 ℃ in advance was added, allowed to stand at room temperature for 1min, and centrifuged for 1 min. The collected DNA solution was stored. And (3) sucking 5 mu L of the extracted plasmid, sending the extracted plasmid to Beijing Huada Gene Limited for sequencing, and storing the successfully sequenced plasmid at the temperature of-20 ℃. Sequencing results show that the T vector obtained by the connection contains a 495bp DNA insert comprising an ORF segment of TaPR1a gene, such as SEQ ID No. 1.

Example 2 construction of wheat transgenic vector pLGY-02 for TaPR1a gene.

Construction of wheat transgenic vector pLGY-02: the plasmid of TaPR1a-T recombinant plasmid and pLGY-02 vector which are correctly sequenced are extracted and double digestion is carried out by using restriction enzymes KpnI + SpeI. The specific enzyme cutting system is as follows: plasmid 1.0. mu.g, KpnI (15U/. mu.L) 1.0. mu.L, SpeI (10U/. mu.L) 1.0. mu.L, 10 XBuffer 2.0. mu.L, ddH₂And O is supplemented to 20 mu L. The enzyme digestion mixture is cut in a metal bath at 37 ℃ for 3-5 h. After enzyme digestion product electrophoresis detection, gel recovery of target gene tabletThe fragment was ligated with the pLGY-02 vector fragment. A connection system: 12. mu.L of the target fragment, 5. mu.L of the pLGY-02 vector fragment, 1.0. mu.L of T4DNA Ligase, 2.0. mu.L of T4DNA Ligase buffer, ddH₂And O is supplemented to 20 mu L. The reagents were mixed well by centrifugation and were either connected at 22 ℃ for at least 1h or in a refrigerator at 4 ℃ overnight. And (3) transforming the ligation product into escherichia coli, carrying out PCR detection, selecting positive bacterial colony shake bacteria, extracting plasmids for double enzyme digestion detection, sending the positive plasmids to a company for sequencing, and screening to obtain a TaPR1a-pLYG-02 recombinant vector.

Example 3 preparation of transgenic plants of TaPR1a overexpressing wheat

The preparation of the agrobacterium-mediated wheat transgenic material is finished by Shandong Jinnan nation biology limited company, the transformation background material is common wheat spring wheat material 'JW 1', and an agrobacterium-mediated wheat immature embryo transformation method is adopted.

Extracting genome DNA by the SDS method: sampling and marking; adding 1 grinding bead into each tube, precooling with liquid nitrogen, leveling, putting into a sample making machine, grinding for 1min at 1100g, taking out, putting into 600 microliter Extraction buffer (100mL, 0.1M Tris-HCl pH 7.5, 100mL, 0.5M EDTA pH 8.0, 125mL, 10% SDS), shaking, and putting into a 65 ℃ water bath for 30 min; taking out, cooling on ice for 15min to room temperature, adding 300 μ L6 MAmmonium Acetate, mixing, placing in 4 deg.C refrigerator for 15min, and centrifuging at 12000g for 15 min; and putting 600 mu L of the supernatant into a 1.5mL centrifuge tube which is already filled with 360 mu L of isopropanol, uniformly mixing, and putting the mixture in a refrigerator at 4 ℃ for precipitation for 15 min. Taking out, centrifuging at 12000g for 15min, and pouring out the supernatant; adding 400 μ L of 75% ethanol, centrifuging at 12000g for 15min, and removing supernatant; repeating the steps once; placing the tube containing the DNA on a superclean bench, opening a cover, and drying by blowing; DNA was redissolved in 100. mu.L of sterile water and allowed to stand at room temperature for about half a day. And (3) carrying out PCR detection on the genome DNA of the transgenic material by using a transgenic vector detection primer to determine a transgenic positive plant.

Example 4 identification of stripe rust and leaf rust resistance of wheat transgenic Material TaPR1a-OE

Purification and propagation of wheat stripe rust: before inoculation, summer spores of a low-temperature stored toxic physiological race CYR32 of wheat stripe rust are activated in warm water at 42 ℃ for 30min, then hydrated, 0.1% Tween-20 is added, activated strains are evenly inoculated on leaves of a wheat stripe rust material JW1 of one heart and one leaf by a smearing method, and after inoculation, water mist is sprayed, the leaves are kept moist for 12-18h under the dark condition of 15 +/-5 ℃ and then are transferred to 15 ℃ for culture. Covering a glass cover and covering gauze, generating a large amount of spore piles on the surfaces of left and right leaves after 12 days of inoculation, collecting summer spores of the rust, scanning and inoculating fresh rust spores to a wheat material with one heart and one leaf to expand and propagate a large amount of rust for a test, collecting the rust spores under the dry condition for later use, storing the rust spores in a silica gel box at 4 ℃ for short term use, and vacuumizing and storing at-20 ℃ for long term storage.

And (3) purifying and propagating the puccinia triticina: before inoculation, summer spores of a low-temperature stored toxicity physiological race THTT of the wheat leaf rust fungus are activated in warm water at 42 ℃ for 30min, then hydrated, 0.1% Tween-20 is added, the activated strains are evenly inoculated on leaves of a heart-leaf wheat leaf rust-sensitive fungus material JW1 by a smearing method, after inoculation, water mist is sprayed, the leaves are kept moist for 12-18h under the dark condition of 25 +/-5 ℃, and then the leaves are transferred to 25 ℃ for culture. Covering a glass cover and covering gauze, generating a large amount of spore piles on the surfaces of left and right leaves after 12 days of inoculation, collecting summer spores of the leaf rust fungi under a dry condition, scanning and inoculating the fresh rust fungi spores to a wheat material with one heart and one leaf, and then, largely propagating the rust fungi for a test, collecting the rust fungi spores under the dry condition for later use, storing the rust fungi spores in a silica gel box at 4 ℃ for short term use, and vacuumizing and storing at-20 ℃ for long term storage.

The identification of the stripe rust and leaf rust resistance of the wheat transgenic material comprises the following steps: after the wheat transgenic material and the wild type thereof are planted for one week, one-leaf one-heart-stage seedlings are obtained. When the first leaf blade is completely unfolded, the purified wheat stripe rust CYR32 and wheat leaf rust physiological race THTT are inoculated by a shaking inoculation method. And (4) placing the inoculated wheat seedlings in a culture room at a proper temperature until the diseases occur. Taking a first leaf of the wheat transgenic material for transgenic identification; and (3) taking a second leaf of the wheat transgene and wild wheat for photographing, and analyzing data of sporulation area percentage by using plant disease phenotype statistics ASSESS software. The result shows that the overexpression of the wheat disease process related protein TaPR1 gene in wheat can obviously improve the resistance level of a plant to wheat stripe rust (figure 1) and wheat leaf rust (figure 2).

Sequence listing

<110> university of agriculture in Hebei

<120> wheat disease process-related protein TaPR1a gene and application thereof in wheat stripe rust and leaf rust resistance

<130> FJ815169.1

<160> 3

<170> PatentIn version 3.5

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<211> 495

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<213> wheat (Triticum aestivum L.)

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gccgtcggcg tgagcgcggt gagctggagc acgaagctgc aggggttcgc ccagagctac 180

gccaaccaga ggatcaacga ctgcaagctc cagcactcgg gcgggcccta cggggagaac 240

atcttctggg ggccggccgg cgcggactgg aaggcggcgg acgcggtgaa gctgtgggtg 300

gacgagaaga aggactacga ctacgggtcc aacacctgcg cgggcgggaa ggtgtgtggg 360

cactacacgc aggtggtgtg gcgcgcgtcg accagcatcg gctgcgctcg cgtcgtctgc 420

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Claims

1. The application of the gene sequence expressed by the over-expression SEQ ID No.1 in improving the disease resistance of common wheat to wheat stripe rust and wheat leaf rust.