CN117947067A

CN117947067A - Wheat ATPase protein, gene and application thereof

Info

Publication number: CN117947067A
Application number: CN202410353492.6A
Authority: CN
Inventors: 刘杰; 康振生; 姚默涵; 钱颖蕊; 胡守俊
Original assignee: Shenzhen Research Institute Of Northwest University Of Agriculture And Forestry Science And Technology
Current assignee: Shenzhen Research Institute Of Northwest University Of Agriculture And Forestry Science And Technology
Priority date: 2024-03-27
Filing date: 2024-03-27
Publication date: 2024-04-30
Anticipated expiration: 2044-03-27
Also published as: CN117947067B

Abstract

The invention discloses a wheat ATPase protein, a gene and application thereof. The amino acid sequence of ATPase protein TaXB is shown in SEQ ID NO:1, has ATPase activity, plays a disease-sensing function in the interaction process of a host plant and the stripe rust, and negatively regulates and controls the resistance of the host plant to the stripe rust. The gene TaXB for coding the ATPase protein TaXB is a susceptibility gene, is infected by the rust bacteria to induce expression in host plants, and has a nucleotide sequence shown in SEQ ID NO: 2. The invention further obtains the transgenic plant edited by the gene TaXB, and verifies that the transgenic interference plant shows resistance to the stripe rust. The invention provides genetic resources and technical support for the subsequent genetic improvement of the strip rust resistant material.

Description

Wheat ATPase protein, gene and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, relates to breeding of plant disease-resistant materials and biological control drug targets in agricultural biotechnology, and in particular relates to a wheat ATPase protein, a coding gene and application thereof.

Background

Wheat stripe rust caused by the specialized form of Puccinia striiformisf (sp. Tritici, pst) is a serious fungal disease in wheat production, the disease year of epidemic can lead to reduced yield of wheat, serious harm and even the failure to harvest. Wheat stripe rust is one of the most serious threats for causing wheat yield loss, has the characteristics of wide occurrence range, high epidemic speed, large harm loss and the like, and seriously threatens the grain production safety.

The control method of wheat stripe rust is mainly chemical control, however, a large amount of unreasonable chemical pesticides used for a long time bring harm to the environment and food safety, and the problems are also gradually attracting general attention, so that disease-resistant breeding is one of the most economical and effective and environment-friendly measures for controlling wheat stripe rust. However, the excavation period of the disease-resistant gene is long, the breeding difficulty of the disease-resistant variety is high, and the toxicity of the stripe rust bacteria is fast changed, so that the disease control is difficult to realize for a long time. Therefore, the creation of a broad-spectrum, durable disease-resistant material is considered as a fundamental approach to control wheat stripe rust.

Plant disease genes, i.e., genes that promote pathogen infestation and colonization, resulting in disease in the host plant. All plant genes promoting infection and affinity can be regarded as Susceptibility (S) genes, and changing the Susceptibility genes of plants can promote plants to generate broad-spectrum and durable disease resistance, and based on the Susceptibility factors of wheat by revealing the pathogenic mechanism of rust, the novel thinking is provided for creating wheat disease resistant materials and breeding materials by using the Susceptibility factors, and the Susceptibility genes have important significance for realizing broad-spectrum and durable disease resistance of wheat breeding.

Disclosure of Invention

Disease-resistant breeding is one of the most effective ways to control wheat stripe rust. The invention aims to research and disclose the wheat susceptible stripe rust factor and provide gene resources for creating stripe rust resistant materials by utilizing the susceptible pathogen factor.

To achieve the above object, the present invention provides a susceptibility gene TaXB to 24. The gene TaXB is infected by the rust bacteria in a host plant to induce expression, and the nucleotide sequence is shown as SEQ ID NO: 2.

Experiments prove that the resistance of a host plant to the rust bacteria is improved by silencing the gene TaXB or silencing a specific gene fragment of the gene TaXB.

Through sequence alignment and specificity analysis, the gene TaXB of the invention has two specific gene fragments, wherein the nucleotide sequence of one specific gene fragment is shown as SEQ ID NO:3, the nucleotide sequence of the other specific gene fragment is shown as SEQ ID NO: 4.

The invention further provides an ATPase protein TaXB. The ATPase protein TaXB is encoded by the susceptibility gene TaXB and has the nucleotide sequence shown in SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.

The ATPase protein TaXB is capable of playing an ATPase function in the interaction process of a host plant and the Rhizopus, and the ATPase protein TaXB has the capacity of decomposing ATP. The ATPase protein TaXB negatively regulates the disease resistance of the host plant by decomposing ATP in the host plant.

The invention also provides an expression vector. The expression vector contains a specific gene fragment of the susceptibility gene TaXB. As described above, gene TaXB has two specific gene fragments, one of which has the nucleotide sequence set forth in SEQ ID NO:3, the nucleotide sequence of the other specific gene fragment is shown as SEQ ID NO: 4.

Also, the invention provides the application of the susceptibility gene TaXB and the coded protein thereof in the cultivation of anti-rust materials.

As a preference for such use, silencing the gene TaXB, or silencing a specific gene fragment of the gene TaXB, can result in increased resistance of the host plant to Rhizoctonia.

The invention also provides a method for cultivating the strip rust resistant material. The method comprises the following steps: creating an editing material by using a CRISPR/Cas9 technology from a host plant ATPase protein gene TaXB to obtain TaXB gene-edited transgenic wheat, wherein the nucleotide sequence of a TaXB gene editing fragment is shown as SEQ ID NO: shown at 5.

For complete and unobjectionable understanding of the technical scheme of the present invention, it should be additionally explained that the ATPase protein coding gene of the present invention is represented by an inclined font "TaXB" and the ATPase protein is represented by a non-inclined font "TaXB". Of course, the meaning and expression of the relevant genes and their encoded proteins can be clearly and completely understood by those skilled in the art from the description of the present invention.

Compared with the prior art, the wheat ATPase protein, the gene and the application thereof have the following beneficial effects or advantages.

1) The invention discloses that ATPase protein TaXB plays a role in the interaction process of the rust and wheat for the first time. The invention lays a theoretical foundation for creating wheat disease-resistant materials by utilizing the ATPase protein TaXB by analyzing the disease-sensitive mechanism, and has important significance for cultivating durable stripe rust-resistant wheat varieties.

2) The invention utilizes a reverse genetics method to analyze the function of an ATPase protein gene TaXB, the gene TaXB is infected and expressed by the stripe rust, a CRISPR/Cas9 gene editing technology is adopted to silence the gene TaXB, the gene TaXB is determined to be a susceptible pathogenic factor, and the ATPase function and the disease-sensing function are exerted in the interaction process of the stripe rust and the wheat.

3) The invention has great significance in improving the disease resistance of wheat. By means of gene engineering technology, wheat ATPase protein gene TaXB and gene TaXB are silenced or knocked out to raise the resistance of wheat to stripe rust.

4) The invention discloses a cultivation method of a wheat stripe rust resistant variety. The method is to obtain the wheat variety with silent gene TaXB by using ATPase protein gene TaXB through CRISPR/Cas9 technology. The transgenic wheat obtained by the method of the invention has been verified to show resistance to the main epidemic race CYR31 of the Rhizoctonia cerealis.

Drawings

FIG. 1 is a preferred technical roadmap for use with the present invention.

FIG. 2 is a graph showing the results of the ATPase activity function assay of ATPase protein TaXB according to an example of the present invention. TaXB24 in the left panel of FIG. 2 represents the normal state of TaXB protein, denatured-TaXB is denatured TaXB protein. The right panel of fig. 2 shows the effect of different metal ions on the enzymatic activity of atpase protein TaXB, NM indicating the absence of metal ions, and other metal ion experimental groups identified in the right panel of fig. 2.

FIG. 3 is a graph showing the results of the VIGS phenotype of ATPase protein gene TaXB according to the example of the present invention. In fig. 3, PDS represents the wheat albino gene, BSMV: gamma represents vector empty control, taXB24-as1 represents the VIGS silencing fragment 1 (nucleotide sequence shown as SEQ ID NO: 3), taXB-as 2 represents the VIGS silencing fragment 2 (nucleotide sequence shown as SEQ ID NO: 4).

FIG. 4 shows the results of the editing site and sequencing of ATPase protein gene TaXB according to the example of the present invention. The upper part of FIG. 4 is a diagram of TaXB gene structure, the numbers 1 and 2 are 2 exons of TaXB gene marked, intron represents introns, exon represents exons, up stream represents upstream gene, down stream represents downstream gene, sgRNA represents guide chain sequence, and PAM represents editing site; FIG. 4 shows the results of the sequencing alignment, WT-A/D representing the A/D chromosome sequence in the wild-type Fielder and taxb a/D representing the A/D chromosome sequence of the editing line.

FIG. 5 is a graph showing the results of the phenotype of the pathogenic type CYR31 of the rust inoculated from transgenic wheat genetically edited in example TaXB of the invention. In FIG. 5, fielder represents the wheat variety Fielder recipient plant and taxb24 represents the editing plant homozygous line.

Detailed Description

The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples.

The experimental methods and the detection methods in the following embodiments are all conventional methods unless otherwise specified; the medicaments and materials are commercially available unless specified; the index data are all conventional measurement methods unless specified.

FIG. 1 shows a preferred technical scheme of the invention, in which wheat is used as an example, for representing a host plant infected with stripe rust. The technical route adopted by other host plants of stripe rust is identical to or slightly different from that of FIG. 1. The embodiment of the invention provides a verification method of the function of ATPase protein TaXB, which comprises the following steps:

s101, verifying the ATPase activity function of the protein TaXB by using an ATPase activity measurement system;

S102, utilizing virus-induced gene silencing technology to specifically and transiently silence TaXB gene, and determining the toxic function of the gene;

S103, obtaining transgenic wheat edited by a gene TaXB and carrying out sequencing analysis on the transgenic wheat;

S104, inoculating the transgenic plant edited by the gene TaXB to the main epidemic race CYR31 of the rust bacteria, identifying the resistance of the transgenic plant to the epidemic race CYR31 of the rust bacteria, and determining the rust resistance of the transgenic wheat with the gene TaXB silenced.

The embodiment of the invention further provides an identification method of the wheat ATPase protein TaXB for the disease sensing function, which comprises the following steps:

the wheat ATPase protein gene TaXB is subjected to specific transient silencing by utilizing a virus-induced gene silencing technology, virus is inoculated in a two-leaf period, whether bleached state appears on wheat leaves after PDS inoculation is detected on 10 th day after inoculation, and if so, the success of virus inoculation is indicated; inoculating fresh spores of wheat stripe rust in the four-leaf period, detecting the number of spore piles on the surface of the leaf at 12 days after inoculation, and checking whether necrosis occurs on the inoculated leaf;

The wheat variety with the ATPase protein gene TaXB silenced is obtained by using the CRISPR/Cas9 technology, and the resistance of the transgenic editing plant to the stripe rust CYR31 is verified.

Example 1

This example provides an isolated cloning assay for wheat ATPase protein TaXB.

The wheat leaves were collected and total RNA of the wheat leaves was extracted using an RNA extraction kit (Beijing Hua Vietnam Biotechnology Co., ltd.).

The total RNA of wheat leaves was reverse transcribed to cDNA first strand using recombinant reverse transcriptase M-MuLV RT (Thermo scientific Co.) under the following reaction conditions: 42 ℃ for 30min;75 ℃ for 5min. Using this as a template, PCR amplification was performed using TaXB24 gene-specific primers TaXB-cDNA-F (5'-ATGGGTTGGCGTTGGCACG-3') and TaXB-cDNA-R (5'-TTACACATCAGTTATCTG-3') to amplify a DNA fragment containing the full-length coding frame of TaXB gene. The PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min;95℃30sec,58℃1min,72℃1min,40 cycles; additional extension at 72℃for 10min.

T-ligation is carried out on the obtained PCR amplified product, the PCR amplified product is constructed into a pMD ™ (Simple) vector (TaKaRa company), escherichia coli DH5 alpha competent cells (Shanghai Weidi biotechnology Co., ltd.) are transformed, positive clones are screened and detected, shaking is carried out overnight, a plasmid is named as T-TaXB, sequencing is carried out by the engineering biotechnology Co., ltd.) to obtain a sequence shown as SEQ ID NO:2, and a nucleotide sequence shown in the following formula.

Example 2

This example provides an ATPase activity functional assay for wheat ATPase protein TaXB.

Primers pMAL-TaXB-F (5'-TCAGAATTCGGATCCATGGGTTGGCGTTGGCACG-3') and pMAL-TaXB-R (5'-GACTCTAGAGGATCC TTACACATCAGTTATCTG-3') were designed based on TaXB gene sequence and PCR amplification was performed to amplify a DNA fragment of the full-length coding frame of TaXB gene containing homologous sequence of pMAL. The PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min;95℃30sec,58℃1min,72℃1min,40 cycles; additional extension at 72℃for 10min.

And constructing the PCR amplification product to a pMAL vector, transforming escherichia coli DH5 alpha competent cells (Shanghai Weidi biotechnology Co., ltd.), screening and detecting positive clones and shaking the bacteria overnight, entrusting the sequencing of the biological technology Co., ltd, retaining plasmids with correct sequencing results for the next experiment, and extracting plasmids to be named pMAL-TaXB.

Positive clones were transformed into E.coli strain BL21 and the medium containing TaXB mL of the monoclonal plaque was picked up and propagated overnight at 37℃and 210 rpm. 1mL of the bacterial liquid was transferred to 100mL of Amp antibiotic broth at 37℃at 210rpm for 2h. When the bacterial liquid concentration reached the logarithmic growth phase, the experimental group inhaled 40. Mu.L of IPTG at a concentration of 1M at 28℃for 8 hours at 150 rpm. After bacterial liquid is collected, an ultrasonic breaker is set as an amplitude transformer 6, the power is 40%, the ultrasonic is started for 3 seconds, and the ultrasonic breaker stops for 5 seconds, and the bacterial liquid is broken on ice for 30 minutes. Centrifuge at 10000rpm for 15min at 4℃and transfer supernatant to another new 50ml collection tube and remove impurities using a 0.45 μm filter followed by bacteria removal using a 0.22 μm filter. Purified fusion proteins were obtained using MBP Trap HP column (GE HEALTHCARE, uppsala, sweden).

TaXB24 ATPase Activity of 24 the ability of Pi to release from ATP was determined spectrophotometrically by ammonium molybdate using an ATPase Activity colorimetric assay kit (catalog number BC0965, solarbio). The quantitative reaction of Pi was carried out at room temperature for 30 minutes and absorbance was measured at 660 nm. Using denatured TaXB protein (Denatured-TaXB) as a control, taXB protein was demonstrated to have atpase activity (fig. 2).

The effect of different metal ions on TaXB enzyme activity was determined in this example, and multiple experimental groups of single metal ions (1 mM Mg ²⁺、Ca²⁺ or Mn ²⁺) and combined metal ions (1 mM Mg ²⁺/Ca²⁺、Mg²⁺/Mn²⁺ or Ca ²⁺/Mn²⁺ or Na ⁺/K⁺; 1:1, v/v) were set, and the results in FIG. 2 indicate that TaXB ATPase activity is dependent on divalent metal ions.

Example 3

This example provides an assay for the effect of transient silenced gene TaXB on virulence of Rhizoctonia cerealis on a specific gene fragment.

Through sequence alignment (http:// blast. Ncbi. Ni. Gov/blast. Cgi) and specificity analysis, the TaXB gene has two gene fragments with higher specificity, and the nucleotide sequence is shown as SEQ ID NO:3 and the nucleotide sequence of the gene fragment 1 is shown as SEQ ID NO:4, and a gene fragment 2 shown in FIG. 4. Based on the two sections of specific gene fragments, a transient silencing vector of the wheat ATPase protein gene TaXB is constructed. For specific procedures for TaXB gene silencing vector construction, reference is made to example 2.

The 3 parts of the Barley Streak Mosaic Virus (BSMV) assembly, alpha, beta, gamma-TaXB, gamma-TaPDS, were linearized separately. The linearized DNA fragment was transcribed into RNA in vitro using RiboMAXTM LARGE SCALE Production System-T7 kit (Promega company) in the reaction system: 37 ℃ for 1h;70 ℃ for 5min.

When the virus is inoculated, the control group adds 10 mu L of alpha, beta and gamma into 200-300 mu L of FES buffer solution, and the liquid is inoculated on two leaves of wheat by a friction inoculation method and rubbed for three times. The experimental groups received α, β, recombinant γ -TaXB or γ -TaPDS in the same manner. Then, the wheat seedlings with the virus are cultured for 10 days at the temperature of 25-28 ℃, and obvious striped chlorosis on the virus-inoculated leaves is observed, so that the virus inoculation is proved to be successful. Wheat seedlings successfully inoculated with the virus are inoculated with wheat stripe rust CYR31, and after 14 days of inoculation culture, the wheat leaves show symptoms, and the leaf morphology of the wheat seedlings after 10 days of inoculation culture and 14 days of inoculation is shown in figure 3.

As shown in FIG. 3, after the wheat ATPase protein gene TaXB is transiently silenced, the spore production of the rust is reduced, and the pathogenicity of the rust is obviously weakened, which indicates that the ATPase protein gene TaXB plays a disease-sensing function in the process of infecting wheat by the rust.

Example 4

This example provides cultivation of ATPase protein gene TaXB, a silencing expression transgenic wheat and a disease resistance identification test thereof.

The TaXB gene silencing vector is led into wheat field acceptor material by agrobacterium induction transformation method, and T0 generation transgenic plant is produced through screening, pre-regenerating, rooting and other steps of calicheamicin resistance callus. The TaXB gene edited transgenic homozygous line was selected for stripe rust resistance identification and CYR31 pathogen inoculation procedure was as described in example 2.

The schematic diagram of the editing sites of the wheat ATPase protein gene TaXB for editing homozygous plant lines and the sequencing result are shown in FIG. 4. The morphology of wheat leaves of the homozygous plant line edited by the wheat ATPase protein gene TaXB and inoculated with the Rhizopus arvensis CYR31 is shown in fig. 5, and as can be seen from fig. 5, the pathogenicity of the Rhizopus arvensis CYR31 to the homozygous plant line edited by the ATPase protein gene TaXB is obviously reduced, and the transgenic plant silenced by the gene TaXB shows resistance to the Rhizopus arvensis CYR 31.

The foregoing embodiments are provided to illustrate the technical scheme of the present invention well, but only the preferred embodiments of the present invention are described, and the scope of the present invention is not limited thereto, and various changes and modifications made by those skilled in the art to the technical scheme of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.

Claims

1.A susceptibility gene TaXB, characterized in that the gene TaXB is infected by rust bacteria to induce expression in host plants, and the nucleotide sequence is shown in SEQ ID NO: 2.

2. The susceptibility gene TaXB of claim 1, wherein silencing the gene TaXB, or silencing a specific gene fragment of the gene TaXB, increases the resistance of a host plant to rust.

3. The susceptibility gene TaXB of claim 1, wherein the nucleotide sequence of the specific gene fragment of the gene TaXB is set forth in SEQ ID NO:3 and/or SEQ ID NO: 4.

4. An atpase protein TaXB, wherein said atpase protein TaXB is encoded by a susceptibility gene TaXB according to claim 1, having the amino acid sequence set forth in SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.

5. The atpase protein TaXB of claim 4, wherein said atpase protein TaXB negatively regulates disease resistance of a host plant during interaction of the host plant with a stripe rust.

6. The atpase protein TaXB of claim 4, wherein said atpase protein TaXB functions in atpase activity during interaction of a host plant with a stripe rust, and wherein said atpase protein TaXB hydrolyzes ATP.

7. The atpase protein TaXB, wherein said atpase protein TaXB negatively regulates host plant disease resistance by promoting atpase activity in a host plant during interaction with a stripe rust.

8. An expression vector comprising the specific gene fragment of susceptibility gene TaXB of claim 1, wherein the nucleotide sequence of the specific gene fragment of gene TaXB is set forth in SEQ ID NO:3 and/or SEQ ID NO: 4.

9. The susceptibility gene TaXB and the application of the coded protein in the cultivation of anti-rust material.

10. The use of claim 9, wherein silencing the gene TaXB, 24, or silencing a specific gene fragment of the gene TaXB, increases the resistance of the host plant to stripe rust.