CN116284286B

CN116284286B - Sugar transporter protein, gene and application of wheat stripe rust

Info

Publication number: CN116284286B
Application number: CN202310236814.4A
Authority: CN
Inventors: 刘杰; 康振生; 潘青林
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2023-03-13
Filing date: 2023-03-13
Publication date: 2025-02-07
Anticipated expiration: 2043-03-13
Also published as: CN116284286A

Abstract

The invention discloses a stripe rust glycotranslocator Pst25662, the amino acid sequence of which is shown as SEQ ID NO. 2. The nucleotide sequence of the sugar transporter expression gene Pst25662 is shown as SEQ ID NO. 1. The sugar transport protein has the sugar and various hexose transport capacities, and the gene Pst25662 is up-regulated to be expressed under the induction of the rust bacteria. The invention utilizes agrobacterium-mediated genetic transformation to obtain transgenic plants with gene Pst25662 silenced, and verifies that transgenic interference plants show resistance to stripe rust. The invention enriches the new cognition of the technicians in the field on the acquisition of the carbon source of the wheat stripe rust, provides ideas for the subsequent research of wheat stripe rust resistant varieties and sustainable regulation and control, and has substantial reference significance on the design of biological control drug targets.

Description

Wheat stripe rust fungus sugar transport 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 stripe rust sugar transport protein, a coding gene and application thereof.

Background

Wheat is the most widely planted grain crop in the world, and about 1/3 of the population worldwide uses wheat as the main grain. Among all biological and abiotic factors which can cause serious yield reduction of wheat, wheat stripe rust is one of the most serious threats for causing yield loss of wheat, and has the characteristics of wide occurrence range, high epidemic speed, large harm loss and the like. In 2002, wheat stripe rust is listed in a national list of insect disease and insect pests. Therefore, perfecting the wheat stripe rust prevention and control measures and ensuring the grain safety production becomes an urgent need to be solved in agricultural production.

Chemical prevention and control are the main mode for preventing and controlling wheat stripe rust at present, but a large amount of unreasonable medicines for a long time not only can increase environmental pollution and increase peasant burden, but also can cause a plurality of problems of chemical medicine residue, disease and insect resistance enhancement and the like, and the natural balance of an agricultural ecological system is destroyed. The cultivation of disease-resistant varieties is one of the most economical, effective and environment-friendly measures for preventing and treating wheat stripe rust, but as stripe rust is frequent in variation of the wheat species, new toxic species are continuously appeared and are popular gradually, and the disease-resistant varieties of the main cultivated wheat are usually overcome in 3-5 years. Therefore, the complete interpretation of the pathogenesis of stripe rust and the creation of a broad-spectrum durable disease-resistant material are considered as the most efficient and direct solution.

Rumex bifidus is a living vegetative parasitic fungus whose survival and breeding is highly dependent on the nutrients taken up from the host. The sugar is an important basic nutrient substance necessary for the growth and development of the rust bacteria of wheat, and the absorption of the rust bacteria to the sugar substance can be controlled to effectively limit the substance and energy supply, so that the growth and development and propagation of the rust bacteria are limited, and the damage to the wheat is further weakened. However, the research on the mechanism of the absorption of the puccinia sugar in the interaction process of wheat and puccinia is relatively few and still not clear. The wheat stripe rust sugar transport protein is relatively conserved in gene evolution, and sugar substances are main forms of pathogenic bacteria in existence, so that a key acquisition mode of the pathogenic bacteria on sugar is defined, a new thought and a breeding material are provided for creating a wheat disease-resistant material by using pathogenic bacteria pathogenic factors, and the wheat stripe rust sugar transport protein has important significance for realizing broad-spectrum and durable disease resistance of wheat breeding.

Disclosure of Invention

The saccharide is the main existence form of pathogenic bacteria carbon source and is essential nutrient for the growth and development of the stripe rust. The invention aims to enrich the new knowledge of the person skilled in the art for obtaining the carbon source of the wheat stripe rust. Specifically, the invention provides a wheat stripe rust sugar transporter gene Pst25662 and a coded protein Pst25662 thereof.

The invention aims at providing a wheat stripe rust sugar transporter Pst25662 and a gene Pst25662 encoding the sugar transporter Pst25662. The invention also provides an application of the sugar transporter Pst25662 and the coding gene thereof in cultivation of wheat stripe rust resistant varieties.

In order to achieve the aim, the technical scheme adopted by the invention is that the wheat stripe rust sugar transport protein Pst25662, the sugar transport protein Pst25662 plays a sugar transport function in the interaction process of stripe rust and wheat, the amino acid sequence of the sugar transport protein Pst25662 is shown as SEQ ID NO.2, and the nucleotide sequence of a gene Pst25662 encoding the sugar transport protein Pst25662 is shown as SEQ ID NO. 1.

Further, the wheat stripe rust sugar transporter gene Pst25662 and the sugar transporter encoded by the wheat stripe rust sugar transporter gene play a sugar transport function in the interaction process of stripe rust and wheat. The specific fragment (the nucleotide sequence is shown as SEQ ID NO. 3) of the gene Pst25662 is silenced, so that the resistance of wheat to stripe rust bacteria can be improved.

The invention also provides an application of the sugar transporter Pst25662 and the coding gene thereof in cultivation of wheat stripe rust resistant varieties.

The invention further provides a wheat disease-resistant breeding method, which comprises the steps of constructing an expression vector containing a wheat stripe rust sugar transporter gene Pst25662 silencing fragment, transferring into a wheat material by using agrobacterium mediation, and obtaining transgenic wheat with the sugar transporter gene Pst25662 silencing, wherein the expression vector contains the Pst25662 gene silencing fragment.

For complete and unobjectionable understanding of the technical scheme of the invention, it is required to supplement that the wheat stripe rust sugar transporter gene is represented by an inclined font "Pst25662" and the wheat stripe rust sugar transporter gene is represented by a non-inclined font "Pst 25662". 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 invention has the following beneficial effects or advantages:

(1) The wheat stripe rust sugar transport protein Pst25662 provided by the invention is a protein with sugar transport function which is reported for the first time in wheat stripe rust, and can transport various monosaccharide substances. Creating a silent plant of the stripe rust sugar transporter Pst25662 by means of host wheat, inhibiting the expression of the sugar transporter when the stripe rust infects the wheat, and inoculating different stripe rust physiological races can lead to the substantial reduction of the spore yield of the silent plant and the increase of immune necrosis cells.

(2) The Pst25662 transgenic plant shows resistance to wheat stripe rust, so that the sugar transport protein Pst25662 can be utilized to create a rust-resistant wheat strain, and an excellent material is provided for cultivation of the wheat rust-resistant wheat strain.

(3) The sugar transport protein gene provided by the invention is relatively conserved among different species, and lays a foundation for creating a disease-resistant wheat variety with durability and broad spectrum. Meanwhile, the method has important reference significance for target research of novel biological control medicines.

Drawings

FIG. 1 is a graph showing the analysis of the expression level of the stripe rust sugar transporter gene Pst25662 in different times after the stripe rust race CYR31 infects wheat, and the data in FIG. 1 are the mean value.+ -. SEM of three biological replicates.

FIG. 2 is a diagram showing the fluorescent localization of the Rhizoctonia cerealis sugar transporter gene Pst25662 provided by the embodiment of the invention, wherein in FIG. 2, free GFP represents GFP protein, pst25662-GFP represents Pst25662 and GFP fusion protein.

FIG. 3 is a graph showing colony growth of sugar transporter gene Pst25662 for a sugar transport function identification test of different carbon sources, wherein Control represents an empty plasmid of pDR195, pst25662 represents a fusion plasmid of pDR195 and Pst25662, maltose represents that the carbon source is maltose, glucose represents that the carbon source is Glucose, sucrose represents that the carbon source is Sucrose, fructose represents that the carbon source is fructose, mannose represents that the carbon source is mannose, mannitol represents that the carbon source is Mannitol, galactose represents that the carbon source is galactose, xylose represents that the carbon source is xylose, and Arabinose represents that the carbon source is arabinose.

FIG. 4 is a graph showing the results of DNA detection of transgenic plants containing the Pst25662 specific silencing fragment, wherein M represents DNAMARKER, H ₂ O represents the water template, WT represents the wild type, PC represents the plasmid, and L19 and L43 represent both RNAi plants 19 and 43.

FIG. 5 is a phenotypic chart of transgenic silent plants inoculated with different Rhizoctonia species CYR32, CYR33, CYR34, respectively, wherein Fielder is a wild type control and L19, L43 represent two transgenic lines of T3 generation 19 and 43.

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 method and the detection method in each embodiment are conventional methods unless otherwise specified, the medicament and the material are commercially available unless otherwise specified, and the index data are conventional measurement methods unless otherwise specified.

Example 1

The embodiment provides analysis tests of different time expression amounts of the stripe rust sugar transporter gene Pst25662 after the stripe rust race CYR31 infects wheat.

1. Acquisition of real-time fluorescent quantitative PCR (polymerase chain reaction) sample of Rumex

Wheat seeds (water source 11) are planted in a flowerpot with the diameter of 10cm, are placed in a light/dark period of 16/8h and are cultured at the temperature of 16 ℃, when wheat seedlings grow to a leaf-core period, fresh wheat stripe rust summer spores are diluted by an electronic fluoridation liquid, 10 mu L of diluted spore liquid is sucked by a liquid-transferring gun, and the diluted spore liquid is evenly smeared and inoculated on wheat leaves. The wheat seedlings after inoculation are continuously cultured under normal illumination conditions after being moisturized for 36 hours in darkness at 15 ℃. Sampling is carried out at the time of 6h, 12h, 18h, 24h, 48h, 72h and 120h respectively after wheat leaf inoculation to serve as real-time fluorescence quantitative PCR samples, each group of sampling selects at least three leaves from different wheat seedlings, and the central area of the inoculated leaf is mainly collected, and the sample is immediately preserved at-80 ℃ for standby.

2. Preparation of Rumex strip templates

The collected sample was used to prepare a cDNA template for real-time fluorescent quantitative detection of Rumex japonicus according to the operation instructions using an RNA extraction kit and vazyme reverse transcription kit (Beijing Hua Vietnam biotechnology Co., ltd.).

3. Detection of real-time fluorescent quantitative PCR

The gene expression condition is analyzed by using the wheat stripe rust elongation factor gene PstEF as an internal reference gene and adopting a relative quantitative method.

The primer sequences of the internal reference genes are as follows:

PstEF-F:TTCGCCGTCCGTGATATGAACAA;

PstEF-R:ATGCGTATCATGGTGGTGGAGTGA。

Pst25662 specific primers were designed by PRIMER PREMIER 5.0.0 software, pst25662 specific primer sequences were:

DL-25662-F:CGTCATTGGCTTAACCGTTCTG;

DL-25662-R:GTCGCCACCACGTTAACTACATT。

Using UltraSYBR One Step RT-qPCRKit fluorescent quantitative PCR kit (Kangji Biotechnology Co., ltd.) according to the instructions, the relevant detection was performed using a Bio-Rad iQ5 real-time fluorescent quantitative PCR instrument. The relative expression levels of the different genes were calculated according to the method of 2 ^-ΔΔCt with the expression level of PstEF as a reference.

As shown in FIG. 1, when the wheat is infected by the Rhizopus arvensis microzyme CYR31 for 12 hours, an air hole lower sac is formed in a cavity below the air hole of the wheat, the Rhizopus arvensis starts to generate a large amount of primary mycelium states, the sugar transporter gene Pst25662 is expressed in a large amount, sufficient preparation is made for sugar substance absorption, the sugar transporter gene Pst25662 expression level is highest at 18 hours, and at the moment, the Rhizopus arvensis is transporting sugar substances from the host body in a large amount. The protein then remains highly expressed, indicating that it has been responsible for harvesting carbohydrate material from wheat tissue and has played an important role.

Example 2

This example provides a localization assay of the sugar transporter Pst25662 in cells.

1. Amplification of the full Length of the Rumex patiens sugar transporter Gene Pst25662

The full-length amplification primer Pst25662-F, pst25662-R was designed using PRIMER PREMIER 5.0.0 software. The primer sequences are as follows:

Pst25662-F:ATGCCTGCCGTAGCCG;

Pst25662-R:TCAAATGTGCTCCAGCTTCTCT。

PCR amplification was performed using the wheat stripe rust cDNA as a template and a 2X TaqMasterMix PCR mixed solution (Kangji Biotechnology Co., ltd.) as a full-length primer. The reaction system is as follows:

The PCR instrument was programmed to pre-denature at 95℃for 5min, denature at 95℃for 15s, anneal at 58℃for 30s, extend at 72℃for 1min, and after 40 cycles, to supplement extension at 72℃for 10 min. After the completion of the PCR procedure, the PCR product was subjected to agarose gel recovery gel detection, the band corresponding to the size of the target gene was recovered, and gel was recovered using the Omega gel recovery kit, and the procedure was performed according to the instructions (Omega Bio-Tek).

The Pst25662 gene fragment obtained by gel recovery was ligated with the vector pMD19-T Simple, transformed into E.coli DH 5. Alpha. Competent (Shanghai Weidi Biotechnology Co., ltd.) and screened for cations. Then the positive seeds are sent to Beijing qingke biotechnology Co Ltd for plasmid sequencing, and plasmids with correct sequencing results are reserved for the next experiment, wherein the plasmids are marked as T-Pst25662.

2. Yeast subcellular localization plasmid construction

GFP primers containing BamHI homology arm sequences were designed based on the sequence characteristics of the multiple cloning site of pDR 195. The primer sequences used were:

195-GFP-F:CGGCCGCGCGGATCCATGGTGAGCAAGGGCG;

195-GFP-R:TCCAAAGCTGGATCCTTACTTGTACAGCTCGTC。

The size-conforming bands were gel recovered using a high fidelity enzyme PRIMER STAR Max Premix (2×), a primer pair 195-GFP-F, 195-GFP-R, a plasmid containing GFP sequences as a template to amplify the GFP sequence containing the BamHI site homology arm of the pDR195 vector. The pDR195 plasmid was digested with BamHI, followed by one-step cloning of the linearized pDR195 plasmid with amplified GFP glue product using ClonExpress II One Step Cloning Kit one-step cloning kit (Nanjinouzan Biotechnology Co., ltd.), construction of pDR195-GFP plasmid, screening Yang Xingzi, and sequencing of correct plasmid retention when pDR195 plasmid already contained GFP sequence.

The Pst25662 primer containing the XhoI homology arm sequence was designed based on the sequence characteristics of the multiple cloning site of the pDR195 vector. The primer sequences are as follows:

195-25662GFP-F:ATATACCCCAGCCTCGAGATGCCTGCCGTAGCCG;

195-25662GFP-R:

CACCATGGATCCCTCGAGAATGTGCTCCAGCTTC。

the size-conforming bands were gel recovered using high fidelity enzyme PRIMER STAR Max Premix (2×), primer pair 195-25662GFP-F, 195-25662GFP-R, and T-Pst25662 plasmids as templates to amplify the Pst25662 sequence containing the homology arm to the XhoI site of the pDR195 vector. One-step cloning was performed as described above using ClonExpress II One Step Cloning Kit one-step cloning kit, and the correct pDR195-25662GFP plasmid was obtained as follows, constructed with the pDR195-GFP plasmid.

3. Yeast subcellular localization

Inoculating single colony of YSL2-1 yeast with YPD (2% maltose) liquid medium, culturing at 30deg.C for 48 hr, transferring yeast mother liquor to the above YPD medium with 2% maltose at 1:100 ratio, shaking until final concentration reaches OD ₆₀₀ of 0.6-0.8, collecting 2mL bacterial liquid, centrifuging at 4500rpm for 5min at room temperature, discarding supernatant, adding 2mL ddH ₂ O, centrifuging at 4500rpm for 5min, discarding supernatant, adding 100 μl LiAc conversion solution (Shanxiprind BioCo., ltd.), slightly suspending, standing for 10min, sequentially adding ssDNA 50 μl, objective plasmid pDR195-GFP or pDR195-25662GFP 3-5 μg, liAc conversion solution 400 μl, mixing, and resuscitating in 30 deg.C shaking table at 200rpm for 30min. The mixture was gently mixed by inversion for 7-8 times every 7.5min by heat-beating at 42℃for 15min, centrifuged at 4500rpm for 5min, and the supernatant was discarded. The cells were resuspended with 500. Mu.L ddH ₂ O, centrifuged at 4500rpm for 5min, the supernatant was discarded, 100. Mu. LddH ₂ O was added for resuspension, and the transformed yeast strain was spread on SD-Ura solid screening medium and cultured for 3 days to obtain positive clones.

The positive transformed strains transferred into pDR195-GFP and pDR195-25662GFP are inoculated into SD-Ura screening culture medium taking 2% maltose as a carbon source, shake culture is carried out for 48 hours at 30 ℃ and 220rpm, the positive transformed strains are transferred into a new screening culture medium for continuous culture at a weight ratio of 1:100, 2mL of bacterial liquid is collected and centrifuged for 1min at 8000rpm at room temperature until the bacterial liquid OD ₆₀₀ value is between 0.5 and 0.7, the supernatant is discarded, 1mL of ddH ₂ O is added for resuspension, a proper amount of yeast liquid is taken for making a slide, and an ultra-high resolution laser confocal microscope LSM880 is used for positioning observation and photographing record. Wherein pDR195-GFP only expresses GFP protein, and pDR195-25662GFP expresses Pst25662 and GFP fusion protein.

As a result, as shown in FIG. 2, only GFP-expressing yeast was observed to express green fluorescence in the whole cells. When the sugar transporter Pst25662 was fused with GFP protein, green fluorescence was observed only on the yeast cell membrane. The localization results indicate that the sugar transporter Pst25662 is localized at the cell membrane, conforming to the membrane localization and transmembrane transport pattern of the sugar transporter.

Example 3

This example provides a validation test for the sugar transport function of the protein Pst 25662.

The Pst25662 primer containing the BamHI homology arm sequence was designed based on the sequence characteristics of the pDR195 multiple cloning site. The pDR195-Pst25662 vector was constructed as in example 2. The primer sequences are as follows:

195-25662-F:CGGCCGCGCGGATCCATGCCTGCCGTAGCCG;

195-25662-R:TCCAAAGCTGGATCCTCAAATGTGCTCCAGC。

the constructed pDR195-Pst25662 and empty vector pDR195 were transformed into yeast sugar transporter deletion mutant YSL2-1 strain, which had knocked out sucrose and hexose transporters, respectively, by a hot-shock transformation method to obtain positive transformants, and the procedure was as in example 2.

The positive transformation strain is inoculated into SD-Ura liquid screening culture medium which takes 2% maltose as a carbon source, and shake culture is carried out at 30 ℃ and 220rpm until the OD ₆₀₀ value of bacterial liquid is between 0.5 and 0.7. 2mL of the bacterial liquid was collected, centrifuged at 8000rpm for 1min at room temperature, and the supernatant was discarded. The cells were resuspended in sterile ddH ₂ O and centrifuged at 8000rpm for 1min, and the cells were washed twice repeatedly. The cell suspension was diluted to the same concentration OD ₆₀₀ =0.6 with sterilized ddH ₂ O, and the shaking was continued at 30 ℃ and 220rpm, and starved for 5 hours. After the completion, the cells were diluted sequentially at 10-fold gradient to obtain cell suspensions of different concentrations, and the cell suspensions were diluted 5 times in total. These cell suspensions were inoculated onto SD-Ura solid screening media containing different sugars as carbon sources (maltose, sucrose, glucose, fructose, galactose, mannose, xylose, arabinose, mannitol) in sequence by pipetting 5. Mu.L each, the sugar concentrations were all 2% (w/v), and colony growth was recorded after 3 days of inversion culture at 30 ℃.

The results are shown in FIG. 3, control represents empty plasmid pDR195 as control group, and Pst25662 represents fusion plasmid of pDR195 and Pst25662 as experimental group. Different carbon sources are added into SD-Ura solid screening culture medium to verify the transport function, wherein maltose is used as a carbon source group to show that yeast is normal in state, and other experimental groups show that the sugar transport protein Pst25662 has the transport capacity of various sugars such as sucrose, glucose, fructose, galactose, mannose and mannitol.

Example 4

The present example provides a test for verifying conservation of Pst25662 gene-silenced transgenic plants to Leptosphaeria Triticum.

1. Construction of transgenic vectors

Sequence comparison is carried out on the sequence ORF region of the stripe rust sugar transporter Pst25662 gene and homologous genes of wheat and wheat stripe rust respectively, pst25662 specific fragment sequences are selected for construction of a transgenic vector, and the nucleotide sequence of the Pst25662 gene silencing fragment (shown as SEQ ID NO: 3) is selected. The selected target fragments are respectively inserted into 2 positions of the PC336 vector by homologous recombination (one fragment is inserted forward and the other fragment is inserted reversely and complementarily, so that a hairpin structure can be formed) to obtain the PC336-25662 plasmid.

The primer sequences containing attB sites are:

attB-25662-F:

GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGCTACATTGCGG GAGT;

attB-25662-R:

GGGGACCACTTTGTACAAGAAAGCTGGGTCCGATGGGATAAGC TAAAAGGGC。

2. transformation of Agrobacterium

Melting EHA105 competent cells stored at-80 ℃ on ice, adding PC336-25662 plasmid with correct sequence, ice-bathing for 30min, liquid nitrogen for 2min,37 ℃ water-bathing for 3min, ice-bathing for 5min, adding a proper amount of liquid LB culture medium, shake culturing for 3h, and coating 150 mu L of transformed bacteria liquid on a solid plate (rifampicin and Kana antibiotics), and culturing in inverted darkness until positive transformants grow.

And transferring the recombinant silencing vector PC336-25662 into a wheat variety Fielder material by using an agrobacterium-mediated wheat transformation technology to obtain an RNAi silencing plant. And (3) detecting DNA of the obtained plant, and reserving the positive plant for subsequent verification.

The results are shown in FIG. 4, and all plants used for transgenic identification are positive plants, and all plants have been successfully transferred into silencing vectors.

3. Phenotype identification of transgenic plants

Two T3 generation transgenic homozygous lines are selected for disease resistance detection, and wheat stripe rust bacteria, namely CYR32, CYR33 and CYR34 are respectively inoculated, and the inoculation method is the same as that of example 1. And carrying out phenotype identification after the wheat leaves are sporulated, and photographing and recording.

As shown in FIG. 5, the yield of the rust race on the silenced plants inoculated with the wheat rust CYR32, CYR33 and CYR34 is obviously reduced. The sugar transport protein Pst25662 provided by the invention has high conservation among different species of the Phlebopus and plays a role as a key sugar transport protein.

In conclusion, the puccinia striolata sugar transport protein Pst25662 plays a key role in the wheat infection process of puccinia striolata, and is mainly characterized by having a plurality of sugar substance transport capacities and always keeping high-level expression in the whole infection stage. The expression of the gene is silenced by constructing a transgenic RNAi plant, so that obvious disease resistance response is generated to various wheat stripe rust bacteria, the gene has higher conservation in the stripe rust bacteria, and the gene has reference significance for creating broad-spectrum disease resistance materials.

The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution 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. The application of the silent stripe rust sugar transporter gene Pst25662 in the cultivation of stripe rust resistant wheat is characterized in that the nucleotide sequence of the sugar transporter gene Pst25662 is shown as SEQ ID NO. 1;

The amino acid sequence of the sugar transporter Pst25662 is shown as SEQ ID NO. 2;

the sugar transporter Pst25662 functions in sugar transport during interaction of the rust with the host plant.