CN114807187A

CN114807187A - Ural chart wheat receptor protein kinase gene TuRLK1 and application thereof

Info

Publication number: CN114807187A
Application number: CN202210479687.6A
Authority: CN
Inventors: 邹声浩; 汤延胜; 唐定中
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2022-05-05
Filing date: 2022-05-05
Publication date: 2022-07-29
Anticipated expiration: 2042-05-05
Also published as: CN114807187B

Abstract

The invention discloses a Ural chart wheat receptor protein kinase geneTuRLK1And applications thereof. The Ural chart wheat receptor protein kinase geneTuRLK1The nucleotide sequence of (A) is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2. Experiments show that the Ural chart wheat receptor protein kinase geneTuRLK1Functional gene for wheat stripe rust resistanceYrU1It is crucial that resistance function is normally exertedExpressed separately in susceptible wheatTuRLK1The resistance level of wheat powdery mildew can be obviously improved; heterologous expression in ArabidopsisTuRLK1And the powdery mildew resistance of arabidopsis can be remarkably improved. Therefore, the Ural chart wheat receptor protein kinase geneTuRLK1Has stronger application potential in plant resistance breeding.

Description

Ural chart wheat receptor protein kinase gene TuRLK1 and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a Ural chart wheat receptor protein kinase geneTuRLKAnd the application of the gene in plant resistance breeding.

Background

Wheat (A), (B)Triticum aestivum) The wheat stripe rust disease is an important grain crop related to the countryside, and the wheat stripe rust disease, the powdery mildew disease and other diseases seriously threaten the growth of wheat and greatly harm the yield and the quality of the wheat. The stripe rust of wheat is caused by stripe rust of wheat (A)Puccinia striiformis f. sp. tritici，Pst) Parasitic fungal diseases caused; wheat powdery mildew is caused by Blumeria necatrix special type (Blumeria graminis f. sp. tritici，Bgt) Causing parasitic fungal diseases. They can all harm wheat and related species, compete with crop leaves for nutrients and hinder photosynthesis. Because the control effect of the applied chemical agents on the pathogenic bacteria is very limited and the application causes pollution to food and ecological environment, the cultivation of wheat varieties with stripe rust and powdery mildew resistance is the most effective coping strategy. It is a current urgent need to clone and study the genes related to high-efficiency resistance carried by wheat and its related species to satisfy the need of resistance breeding.

The Ural chart wheat is a wild ancestral species of the genome A of the common wheat, and compared with the other two ancestral species of the common wheat (aegilops tauschii and aegilops tauschii), the characteristics of the plant morphology, the wheat ear development and the like are more similar to those of the common wheat. The whole genome sequence of the wheat material G1812 of the Ural chart is sequenced and spliced into 7 complete chromosomes, and a large number of functional genes are annotated. The Wularch diagram wheat material PI428309 has excellent resistance to a plurality of strains of Triticum aestivum and Triticum aestivum. Through map-based cloning and complementation verification, we have cloned and verified several resistance function genes from PI428309 in previous work. Wherein Pm60 is a typical CC-NBS-LRR protein and endows PI428309 with powdery mildew resistance function; YrU1 is an R protein with ankyrin structural domain at N-terminal and WRKY structure at C-terminal, and determines the stripe rust resistance of PI 428309. These findings lay the foundation for the full utilization of PI428309 in resistance breeding.

However, the excavation and research of wheat disease-resistant genes are still in the beginning stage at present, the source-resistant genes which can be directly provided for resistance breeding are not abundant enough, and the understanding of resistance mechanisms is more rough and shallow. Plants often have multiple levels of defense patterns against pathogenic bacteria, with interactive effects between the different patterns involving a large number of complex resistance-associated genes. These conditions are extremely unfavorable for the scientific and effective utilization of resistance genes to develop broad-spectrum durable resistant wheat varieties. Therefore, in order to deeply understand the mechanism of the plant for resisting parasitic pathogenic fungi and further cultivate excellent resistant varieties, the cloning and the exploration of more wheat resistance genes are urgently needed.

Against these backgrounds, the present invention inoculates wheat stripe rust fungus on wularch wheat material PI428309PstThe samples before and after the analysis of transcriptome sequencing find and verify one receptor-like protein kinase geneTuRLK1After inoculation with pathogenic bacteria, the transcript levels rapidly increase. Gene silencing experiments prove that the receptor-like protein kinase geneTuRLK1Wheat stripe rust resistance gene for Wularch wheat material PI428309YrU1Essential for the development of resistance. Moreover, further over-expression experiments also prove that the receptor-like protein kinase geneTuRLK1Has important functions in wheat powdery mildew resistance and arabidopsis powdery mildew resistance. The work of the invention provides a new high-efficiency resistance source for breeding practice so as to improve the resistance of common wheat to pathogenic bacteria on the premise of ensuring the yield and quality of the common wheat. Through the application of protein kinase gene to Ural chart wheat receptorTuRLK1The functional research can further understand the mechanism of the plant for resisting parasitic pathogenic fungi.

Disclosure of Invention

The invention aims to provide a Ural chart wheat receptor protein kinase geneTuRLK1And the application thereof, provides a novel and efficient antigen gene for wheat stripe rust and powdery mildew resistance breeding, so as to culture an excellent wheat resistance variety and deepen the disease resistance of plantsThe understanding of the principle is made.

In order to solve the technical problems, the invention adopts the following technical scheme:

the invention firstly provides a Ural chart wheat receptor protein kinase geneTuRLK1The said Ural chart wheat receptor protein kinase geneTuRLK1The nucleotide sequence of the coding region is shown as SEQ ID NO. 1.

The invention also provides a protein kinase gene of the Wularch wheat receptorTuRLK1The coded protein has an amino acid sequence shown as SEQ ID NO. 2.

The invention also provides a wheat receptor protein kinase gene containing the Ural chartTuRLK1The recombinant vector of (1).

The invention also provides the Ural chart wheat receptor protein kinase geneTuRLK1Application in the construction of stripe rust resistant wheat varieties.

The invention also provides the Ural chart wheat receptor protein kinase geneTuRLK1Application in constructing powdery mildew resistant wheat varieties.

The invention also provides the Ural chart wheat receptor protein kinase geneTuRLK1Application in constructing powdery mildew resistant arabidopsis material.

The invention has the beneficial effects that:

the invention clones receptor-like protein kinase gene in Ural chart wheat PI428309TuRLK1It can not only regulate and control the resistance gene of Wularg pattern wheat PI428309 for stripe rustYrU1To pairPstThe resistance of CYR33 can also play a role in resisting powdery mildew in common wheat, which indicates that the receptor protein kinase geneTuRLK1Has wide disease resistance in wheat plants. In addition, Ural map wheat receptor protein kinase geneTuRLK1The gene can also play a powdery mildew resistance function in arabidopsis thaliana, which shows that the gene can also play a resistance role in heterologous plants. It can be seen that the receptor-like protein kinase gene originated from Wularch wheat PI428309TuRLK1Having resistance to wheat powdery mildew, stripe rust and inThe resistance function in heterologous plants has important application value in the field of plant disease resistance breeding.

Drawings

FIG. 1: the sequence of Ural diagram wheat PI428309 receptor protein kinase TuRLK1 is compared with Ural diagram wheat G1812 receptor protein kinase TRIUR3_ 02522.

FIG. 2: silencingTuRLK1Wheat PI428309 pairs causing Wularg patternPst CYR33Is significantly reduced.

FIG. 3: BSMV:GFPand BSMV:TuRLK1inoculating stripe rust fungus after infecting Wularg pattern wheat PI428309Pst CYR33Histological observation of fungal growth. a, uller map wheat PI428309 is modified by BSMV:GFPand BSMV:TuRLK1infecting and inoculating with stripe rust fungusPstObserving the structural morphology change of 2 dpi, 3 dpi and 5 dpi of the puccinia striiformis growing on the wheat leaves after CYR33 by using Wheat Germ Agglutinin (WGA) staining, SSV (air sacs), PH (primary infection hypha), HMC (haustorium cells) and H (haustorium); b, after 2 dpi of rust streaks are inoculated, counting BSMV, GFP and BSMV:TuRLK1the number of Hyphal Branches (HB), Haustorium Mother Cells (HMC) and haustorium number (H) of the rust streaks after treatment; c, after inoculating 3 dpi to the rust streaks, counting the length of hypha infected by the rust streaks (P)<0.001); d, after 5 dpi of rust streaky fungus inoculation, counting the hypha infection area (P)< 0.05）。

FIG. 4: transient expression of single cell on common wheat Fielder leafTuRLK1Can remarkably reduce the inoculationBgtSucker index after E09.

FIG. 5: over-expressionTuRLK1Enhancing the resistance of arabidopsis to powdery mildew. a, phenotype of Arabidopsis thaliana material grown for four weeks after inoculation with powdery mildew for 7 days, WT isArabidopsis thaliana accession Col-0，pad4For the control arabidopsis mutants of the disease,TuRLK1-7 ^# andTuRLK1-10 ^# for overexpressionTuRLK1 Transgenic T of Arabidopsis thaliana ₂ Generation plant, Bar = 8 mm; b, inoculating 7 days of arabidopsis thaliana leaf trypan blue staining results, wherein Bars = 100 mu m; c, counting the conidiophores of the arabidopsis thaliana growing around 5 days after quantitative inoculation of powdery mildewFruit; d, trypan blue staining results of 5 days inoculated arabidopsis leaves, Bars = 100 μm.

Detailed Description

The following examples are presented to illustrate and explain the present invention and are not intended to limit the scope of the invention.

The invention inoculates wheat stripe rust fungus to Wularch wheat material PI428309PstTranscriptome sequencing analysis is carried out on samples 36 h before and after CY33, and one receptor-like protein kinase gene is foundTuRLK1The expression level is obviously increased after the inoculation of pathogenic bacteria. By targeting receptor-like protein kinase genesTuRLK1Silencing, transient expression and stable transformation are carried out, and the invention proves that the receptor-like protein kinase geneTuRLK1Plays an important role in wheat stripe rust resistance, wheat powdery mildew resistance and arabidopsis powdery mildew resistance. For the convenience of the reader to understand the contents and objects of the present invention, the detailed technical implementation steps of the present invention will be described in detail by the following examples. The technical means described in the examples are conventional means familiar to the person skilled in the art. The disease or infection resistance described in the following examples refers to stripe rust and powdery mildew; stripe rust disease is stripe rust disease of wheat, which is caused by stripe rust of wheat (A)Puccinia striiformis f. sp. tritici，Pst) Caused by this, in the following, if there is no special mark, the rust (I) tritici strains CY33 (which is popular in northwest China)PstCYR 33). The powdery mildew refers to wheat powdery mildew and arabidopsis thaliana powdery mildew, which are respectively prepared from wheat powdery mildew (A), (B), (C) and (C)Blumeria graminis f. sp. tritici，Bgt) And Arabidopsis thaliana powdery mildew (Golovinomyces cichoracearum) And is caused by this. Hereinafter, if not specifically identified, wheat powdery mildew is specifically wheat powdery mildew strain E09 (popular in northern China)BgtE09) In that respect The Arabidopsis thaliana described herein is the Arabidopsis thaliana Columbia (Clo) ecotype.

Example 1 Ural diagram wheat Material PI428309 receptor-like protein kinase GeneTuRLK1Cloning of (2)

Inoculating wheat stripe rust fungus after the Wularg pattern wheat PI428309 grows to one leaf and one heartPstCY33, inoculated at two time points, 0 h and 36 h after inoculationPuccinia striiformis (Fr.) KuntzePstWheat leaves from the Ural chart after CY33 were sampled for transcriptome sequencing. All the differentially expressed genes determined in the transcriptome were analyzed with reference to the genomic sequence of wheat G1812 in the Ural chart. Ural map wheat G1812 receptor protein kinase geneTRIUR3_02522 is a receptor-like protein kinase gene of the LRR-RLK type. Inoculation according to the results of transcriptome analysisPstAfter the time of CY33, the first,TRIUR3homologous gene of _02522 in Ural chart wheat PI428309TuRLK1The transcription level of (A) is greatly increased. Preliminarily obtaining receptor-like protein kinase genes according to a transcriptome splicing database of Wulare diagram wheat PI428309TuRLK1The mRNA sequence of (1). To verify the true presence in Ural diagram wheat PI428309TuRLK1full-Length Gene, design of specific primers: (TuRLK1-F andTuRLK1-R), reverse transcription by Promega kit and high fidelity amplification by KOD-Fx DNA polymerase (TOYOBO)TuRLK1The gene sequence of (1). Obtaining using online code region prediction softwareTuRLK1The coding region sequence of (A) is shown as SEQ ID NO.1, and the amino acid sequence of the coding protein is shown as SEQ ID NO. 2. Specific primerTuRLK1-F andTuRLK1-the nucleotide sequence of R is as follows:

TuRLK1-F：5’-CGCACGGGGCTCTAGAGATGACTA-3’，

TuRLK1-R：5’-AGCAGCGAAGCAAACAAGGGTCA-3’。

as shown in FIG. 1, the Ural diagram wheat PI428309 type receptor protein kinase TuRLK1 has different amino acid sequence deletions in LRR (Leucine-Rich Repeat Region) Region compared with the Ural diagram wheat G1812 type receptor protein kinase TRIUR3_02522 protein sequence.

Example 2TuRLK1Regulating and controlling Ural pattern wheat PI428309YrU1Mediated stripe rust resistance

Taking cDNA of Wulare map wheat PI428309 leaf as a template and primer gamma-TuRLK1-F and γ -TuRLK1And (4) amplifying the-R to obtain a PCR product. Carrying out enzyme digestion on a Barley stripe viral RNA gamma (BSMV) plant viral vector by using a restriction enzyme Nhe I to obtain a linearized plasmid carrying an enzyme digestion siteAnd constructing the PCR product into a silent vector BSMV by using a homologous recombination Kit Vazyme Clon express II One Step Cloning Kit C112 to obtain the silent vector BSMV: TuRLK1. Viral vector BSMV with incorporated GFP fragment: GFPas a control, reference is made to the method provided by [ Holzberg S, Brosio P, Gross C, Pogue GP. 2002, Barley stripe virus-induced gene cloning in a monocot Plant, Plant J30: 315- & 327 ], which infects one-leaf, one-heart-stage Ural map wheat PI428309, and after virus infection, the fourth leaf is taken and inoculated with Acremonium stripe CY33 ex vivo and testedTuRLK1The expression level of (a); the material G1812 of Ural chart for susceptibility to stripe rust CY33 is used as negative control, and Ural chart wheat PI428309 (CK) which is not infected by virus is used as positive control. Primer gamma-TuRLK1-F and γ -TuRLK1The nucleotide sequence of R is as follows:

γ-TuRLK1-F：5’-ttttttttttttttagctagcggtggtagaagtggccagattg-3’，

γ-TuRLK1-R：5’-gattcttcttccgttgctagcgtgtgcaggtgtctgagcacg-3’。

as a result, it was found (fig. 2) that, relative to the control group BSMV:GFP，BSMV: TuRLK1group ofTuRLK1Expression level down regulation, silencingTuRLK1Wheat PI428309 pairs capable of causing stripe rust resistance Ural chartPstCY33 is infected. This experiment strongly provesTuRLK1Participating in regulation and control of Ural chart wheat PI428309YrU1Mediated stripe rust resistance.

Sampling is carried out at different time stages after the rust streaks are inoculated, the fungus structure is dyed by Wheat Germ Agglutinin (WGA), and indexes of 2 dpi, 3 dpi and 5 dpi rust spore germination conditions, hypha length, hypha infection area size and the like are counted. As a result, it was found (fig. 3) that, on day 2 after inoculation, BSMV:TuRLK1group HB (hyphal branching) number vs control BSMV:GFPa plurality of about 2.26 and 1.90, respectively; BSMV:TuRLK1the number of HMCs (haustorium blasts) of the group compared to the control BMSV:GFPmore, about 2.08 and 1.82, respectively; BSMV:TuRLK1the number of H (haustorium) of the group was greater than the control BSMV:GFPa plurality of about 1.24 and 1.04, respectively; day 3 after inoculation, BSMV:TuRLK1hypha of groupGermination length ratio control group BSMV:GFPthe length is respectively about 113.92 μm and 87.05 μm, and the t test shows that the difference is very significant; day 5 after inoculation, BSMV:TuRLK1hyphal infestation area of group compared to control BSMV:GFPa significant increase, respectively about 17242.12 μm ² And 11325.74 μm ² There were very significant differences. These results show that whenTuRLK1When adjusted downward, the growth of the rust is favored, which is illustrated from the sideTuRLK1Plays an important role in the resistance of Puccinia striiformis CY 33.

Example 3 common wheat overexpressionTuRLK1Can enhance the resistance of the plant to powdery mildew

Reference is made to the methods provided [ Shen Q-H, Saijo Y, Mauch S, Biskup C, Bieri S, Keller B, Seki H, Ü lker B, Somsich IE, Schulze-Lefert P.2007 ] nucleic Activity of MLA Immune Receptors Links isocyanate-Specific and basic Disease-Resistance responses. Science 315: 1098-TuRLK1The powdery mildew resistance function of the compound is verified. On the leaf of common wheat Fielder infected with powdery mildew, gene gun mediated single cell transient expressionTuRLK1(will be with gateway system)TuRLK1The coding region is connected with an intermediate vector pDONR207 and then transferred into a final vector pUBI-GW for amplificationTuRLK1The coding region of (A) using a primer ofTuRLK1-attB-F andTuRLK1-attB-R), inoculation 4 h after expressionBgtE09, GUS staining after 48 h, and counting the haustorium index. Primer and method for producing the sameTuRLK1-attB-F andTuRLK1the nucleotide sequence of attB-R is as follows:

TuRLK1-attB-F：5’-ggggacaagtttgtacaaaaaagcaggcttcatggcgaggctgctgctcggg-3’，

TuRLK1-attB-R：5’-ggggaccactttgtacaagaaagctgggtcctaggagatgaccgcctccgcccac-3’。

as a result, it was found (FIG. 4) that overexpression was carried outTuRLK1Compared with control treatment (over-expression of PGY protein), the method can remarkably reduce the powdery mildew haustorium index on the infected wheat Fielder leaves and improve the powdery mildew resistance.

Example 4 heterologous overexpressionTuRLK1Enhancing white color of Arabidopsis thalianaPowdery mildew resistance

Constructing receptor-like protein kinase gene driven by constitutive CaMV 35S promoter by using pEARLEY201 plasmid as basic vectorTuRLK1The plant expression vector pEARLEY201-TuRLK1(ii) a Agrobacterium GV3101 strain is transferred by freeze thawing method and Arabidopsis thaliana is genetically transformed by inflorescence dip-dyeing method. Screening positive plants by using herbicide Basta, and detecting the contents of the positive plantsTuRLK1The expression level of (a). Powdery mildew disease mutant of arabidopsis thalianapad4Is a negative control.

To clarify the heterologous overexpression of receptor-like protein kinase genesTuRLK1The effect on the resistance function of arabidopsis powdery mildew, and an inoculation powdery mildew experiment is performed on 4-week-old transgenic arabidopsis thaliana. As a result, it was found (FIG. 5) that conidia were densely distributed on the leaf surface of wild type Arabidopsis thaliana 7 days after inoculation, and there was no cell death, whileTuRLK1The powdery mildew infection positions of the leaves of the transgenic lines are all generated by cell death; the observation of trypan blue staining also revealed that,TuRLK1the conidiophores and hyphae of powdery mildew on the leaves of the transgenic plants are obviously reduced, and cell death at multiple positions can be observed; quantitative inoculation of powdery mildew and subsequent analysis also revealed that T at 4 weeks of age ₂ On the generation-positive transgenic plants, the number of conidiophores (50 single spore colonies counted per material) produced by single powdery mildew spore germination was significantly less than that of wild-type arabidopsis (fig. 5). From the above data, it can be seen that when powdery mildew is infected, heterologous expression is performedTuRLK1Can induce the leaf blade of arabidopsis thaliana to generate cell death, thereby inhibiting the invasion and growth of pathogenic bacteria and enhancing the powdery mildew resistance of arabidopsis thaliana.

The foregoing has outlined rather broadly the present invention in terms of a general description and a specific embodiment. It will be readily apparent to those skilled in the art that certain changes and modifications may be made therein without departing from the spirit of the invention, and it is intended to claim all such modifications as fall within the scope of the invention.

SEQUENCE LISTING

<110> Fujian agriculture and forestry university

<120> Ural chart wheat receptor protein kinase gene TuRLK1 and application thereof

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<400> 8

ggggaccact ttgtacaaga aagctgggtc ctaggagatg accgcctccg cccac 55

Claims

1. Ural chart wheat receptor protein kinase geneTuRLK1The method is characterized in that: the Ural chart wheat receptor protein kinase geneTuRLK1The nucleotide sequence of (A) is shown in SEQ ID NO. 1.

2. The Ural chart wheat receptor protein kinase gene as claimed in claim 1TuRLK1The encoded protein characterized by: the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. A protein kinase gene containing the Ural chart wheat receptor protein of claim 1TuRLK1The recombinant vector of (1).

4. As claimed in claim 1The Ural chart wheat receptor protein kinase geneTuRLK1Application in the construction of stripe rust resistant wheat varieties.

5. The Ural chart wheat receptor protein kinase gene as claimed in claim 1TuRLK1Application in constructing powdery mildew resistant wheat varieties.

6. The Ural chart wheat receptor protein kinase gene as claimed in claim 1TuRLK1Application in constructing powdery mildew resistant arabidopsis material.