CN114807187B

CN114807187B - Ula drawing wheat receptor protein kinase gene TuRLK1 and application thereof

Info

Publication number: CN114807187B
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: 2023-08-18
Anticipated expiration: 2042-05-05
Also published as: CN114807187A

Abstract

The invention discloses a wheat receptor protein kinase gene of uracratiaTuRLK1And applications thereof. Ula drawing wheat receptor protein kinase geneTuRLK1The nucleotide sequence of the polypeptide is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. Experiments show that the urapids wheat receptor protein kinase geneTuRLK1Not only for wheat stripe rust resistance functional geneYrU1It is important that the resistance function is normally exerted and the expression is carried out in the infected wheat aloneTuRLK1The resistance level of the wheat powdery mildew can be obviously improved; heterologous expression in Arabidopsis thalianaTuRLK1Can also remarkably improve powdery mildew resistance of arabidopsis. Thus, urapida wheat receptor protein kinase geneTuRLK1Has stronger application potential in plant resistance breeding.

Description

Ula drawing wheat receptor protein kinase gene TuRLK1 and application thereof

Technical Field

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

Background

Wheat @Triticum aestivum) Is an important grain crop related to national folk life, and diseases such as wheat stripe rust, powdery mildew and the like seriously threaten the growth of wheat, and greatly endanger the yield and quality of the wheat. Wheat stripe rust is prepared from wheat stripe rust bacteriaPuccinia striiformis f. sp. tritici，Pst) Parasitic fungal disease initiated; the wheat powdery mildew is characterized by the specific wheat of the powdery mildew of brusselsBlumeria graminis f. sp. tritici，Bgt) Parasitic fungal diseases caused. They can harm wheat and its kindred species and worksLeaves compete for nutrients and hinder photosynthesis. Since the application of chemical agents has very limited control effects on the pathogenic bacteria and pollutes grain and ecological environment, cultivation of wheat varieties with stripe rust and powdery mildew resistance is the most effective countermeasure. The cloning and research of high-efficiency resistance related genes carried by wheat and related species thereof to meet the requirement of resistance breeding are urgent matters at present.

The urapidotia wheat is a wild ancestor species of the A genome of common wheat, and the plant morphology and other characteristics of the urapidotia wheat are more similar to those of the common wheat compared with the other two ancestor species (aegilops similis and aegilops crudus) of the common wheat. Wherein, the whole genome sequence of the urapidotian wheat material G1812 is already sequenced and spliced into 7 complete chromosomes, which annotate a large number of functional genes. The uralensis wheat material PI428309 has excellent resistance to a plurality of wheat stripe rust strains and wheat powdery mildew strains. Through map cloning and complementation verification, we have cloned and verified several resistance functional genes from PI428309 in previous work. Wherein, pm60 is a typical CC-NBS-LRR protein, which endows PI428309 powdery mildew resistance function; yrU1 is an R protein with an ankyrin domain at the N-terminus and a WRKY structure at the C-terminus, and determines the stripe rust resistance of PI 428309. These findings lay the foundation for the full use of PI428309 in resistance breeding.

However, the current excavation and research of wheat disease resistance genes is still in a starting stage, but the source resistance genes which can be directly provided for resistance breeding are not abundant enough, and the understanding of resistance mechanisms is thicker and shallower. Plants often have multiple levels of defense patterns against pathogens, with different patterns of interaction involving a large number of complex resistance-related genes. These conditions are extremely detrimental to the scientific and efficient use of resistance genes to develop wheat varieties with a broad spectrum of durable resistance. Therefore, in order to understand the mechanism of plant resistance against parasitic pathogenic fungi in depth and further to develop superior resistant varieties, there is also an urgent need to clone and explore more wheat resistance genes.

Under the background, the present inventionThe invention inoculates the uracratia on the uracratia wheat material PI428309PstTranscriptome sequencing analysis is carried out on the front and back samples, and one receptor-like protein kinase gene is found and verifiedTuRLK1After inoculation with pathogenic bacteria, transcript levels rise rapidly. Gene silencing experiments prove that the receptor-like protein kinase geneTuRLK1Wheat stripe rust resistance gene for uracrat wheat material PI428309YrU1It is essential to exert a resistance function. Furthermore, further over-expression experiments also prove that the receptor-like protein kinase geneTuRLK1Has important functions in the aspect of wheat powdery mildew resistance and arabidopsis powdery mildew resistance. The invention provides a new high-efficiency antigen for breeding practice, so that the resistance of common wheat to pathogenic bacteria is improved on the premise of ensuring the yield and quality of the common wheat. Through the gene of the protein kinase of the wheat receptor of the urapidotiTuRLK1The functional research is carried out to further understand the mechanism of the plant for resisting parasitic pathogenic fungi.

Disclosure of Invention

The invention aims to provide a uracratia wheat receptor protein kinase geneTuRLK1And the application thereof provides novel and efficient source resistance genes for wheat stripe rust and powdery mildew resistance breeding so as to cultivate excellent wheat resistant varieties and deepen the understanding of plant disease resistance mechanisms.

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

the invention firstly provides a uracratia wheat receptor protein kinase geneTuRLK1The urapidotian 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 uracratia 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 uralensis graphTuRLK1Is a recombinant vector of (a).

The invention also provides the black-bone LalaEr-diagram wheat receptor protein kinase geneTuRLK1The application in constructing stripe rust resistant wheat varieties.

The invention also provides the uralensis wheat receptor protein kinase geneTuRLK1The application in constructing powdery mildew resistant wheat varieties.

The invention also provides the uralensis wheat receptor protein kinase geneTuRLK1The application in construction of powdery mildew resistant Arabidopsis materials.

The invention has the beneficial effects that:

the invention clones in Ula diagram wheat PI428309 to obtain receptor-like protein kinase geneTuRLK1It can regulate and control the stripe rust resistance gene in Ula drawing wheat PI428309YrU1For a pair ofPstThe resistance of CYR33 can also exert powdery mildew resistance function in common wheat bodies, which shows that the receptor protein kinase genesTuRLK1There is a broad spectrum of disease resistance in wheat plants. In addition, urapida wheat receptor protein kinase geneTuRLK1The powdery mildew resistance function can be exerted in the arabidopsis thaliana body, which shows that the gene can exert the resistance function in heterologous plants. It can be seen that the receptor-like protein kinase gene derived from urapidotia wheat PI428309TuRLK1Has the resistance function in wheat powdery mildew and stripe rust resistance and in heterologous plants, and has important application value in the field of plant disease resistance breeding.

Drawings

Fig. 1: sequence comparison of urapidine wheat PI428309 receptor protein kinase TuRLK1 with urapidine wheat G1812 receptor protein kinase triur3_ 02522.

Fig. 2: silencingTuRLK1Causing urapidotian wheat PI428309 pairPst CYR33Is significantly reduced.

Fig. 3: BSMV:GFPand BSMV:TuRLK1inoculation of Rumex strigosus after infection of Ula diagram wheat PI428309Pst CYR33Is a histological observation of fungal growth. a, uralensis wheat PI428309 is BSMV:GFPand BSMV:TuRLK1infection with rust bacteriaPstAfter CYR33, the strip rust is observed by Wheat Germ Agglutinin (WGA) stainingStructural morphology changes of the fungus after growing 2 dpi, 3 dpi and 5 dpi on wheat leaves, SSV (stomatal inferior sac), PH (primary infection hypha), HMC (aspirator parent cell) and H (aspirator); b, after inoculating 2 dpi of the stripe rust, counting BSMV (binary sequence of GFP and BSMV):TuRLK1after treatment, the number of Hyphal Branches (HB), haustorium blasts (HMC) and haustorium numbers (H) of the rust; c, after inoculating 3 dpi of the rust, counting the length of the rust-infected hypha (P<0.001 A) is provided; d, after inoculating 5 dpi of Rumex striolatus, the hypha infection area (P< 0.05）。

Fig. 4: transient expression of single cells on leaves of the wheat FielderTuRLK1Can significantly reduce its inoculationBgtAspirator index after E09.

Fig. 5: overexpression ofTuRLK1Resistance of arabidopsis thaliana to powdery mildew is enhanced. a, phenotype of four-week-growing Arabidopsis materials inoculated with powdery mildew for 7 days, wherein WT isArabidopsis thaliana accession Col-0，pad4For the disease-sensitive control of the Arabidopsis mutant,TuRLK1-7 ^# andTuRLK1-10 ^# for overexpression ofTuRLK1 Arabidopsis transgenic T ₂ Plants were replaced, bar=8 mm; b, inoculating an arabidopsis thaliana leaf trypan blue dyeing result of 7 days, wherein bars=100 mu m; c, quantitatively inoculating the four-week-growing arabidopsis materials with the conidiophore statistical result after 5 days of powdery mildew; d, after 5 days of inoculation, the arabidopsis leaves are dyed with trypan blue, wherein bars=100 mu m.

Detailed Description

The following examples are intended to illustrate, but not limit the scope of the invention.

The invention inoculates the uracratia on the uracratia wheat material PI428309PstTranscriptome sequencing analysis is carried out on samples of CY33, front and rear 36 and h, and one receptor-like protein kinase gene is foundTuRLK1The expression level is significantly increased after inoculation with pathogenic bacteria. By gene for receptor-like protein kinaseTuRLK1Silencing, 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 convenience ofThe reader will understand the contents and objects of the present invention, and will describe the specific technical implementation steps of the present invention in detail by the following examples. The technical means described in the examples are conventional means familiar to those skilled in the art. Disease resistance or susceptibility, i.e., stripe rust, powdery mildew, as described in the examples below; the stripe rust is wheat stripe rust and is prepared from wheat stripe rust bacteriaPuccinia striiformis f. sp. tritici，Pst) The strain CY33 of the wheat rust bacteria which is popular in northwest China is specifically caused by the fact that the wheat rust bacteria without special marks are hereinafterPstCYR 33). The powdery mildew refers to wheat powdery mildew and arabidopsis powdery mildew, and the wheat powdery mildew and the arabidopsis powdery mildew are respectively prepared from the wheat powdery mildewBlumeria graminis f. sp. tritici，Bgt) And Arabidopsis thaliana powdery mildewGolovinomyces cichoracearum) Caused by the method. In the following, if no special mark exists, the wheat powdery mildew is specifically the wheat powdery mildew strain E09 which is popular in the north of ChinaBgtE09 A kind of electronic device. The Arabidopsis thaliana is an Arabidopsis thaliana Columbia (Clo) ecotype.

Example 1 Ula drawing wheat material PI428309 receptor protein kinase geneTuRLK1Is cloned from (A)

Inoculating wheat stripe rust after growth of Ural diagram wheat PI428309 to one leaf and one heart periodPstCY33, inoculated with rust on wheat at two time points, 0 h and 36 h, respectively, after inoculationPstSamples of uralensis wheat leaves after CY33 were taken and transcriptome sequenced. All the differentially expressed genes measured in the transcriptome were analyzed with respect to the urapidotian wheat G1812 genomic sequence. Ula diagram wheat G1812 receptor protein kinase geneTRIUR302522 is a receptor-like protein kinase gene of the LRR-RLK type. Based on the results of transcriptome analysis, vaccinationPstAfter the period of CY33, the process is completed,TRIUR3homologous gene of 02522 in Ula drawing wheat PI428309TuRLK1Is greatly increased. According to the transcriptome splice database of the uralensis diagram wheat PI428309, the receptor-like protein kinase gene is preliminarily obtainedTuRLK1Is a sequence of mRNA of (A) in a host cell. To verify the true presence in urapidotian wheat PI428309TuRLK1Designing specific primer for full-length geneTuRLK1-F andTuRLK1-R) by Promega kit reverse transcription and KOD-Fx DNA polymerase (TOYOBO) high-fidelity amplificationTuRLK1Is a gene sequence of (a). Obtaining by using online coding region prediction softwareTuRLK1The coding region sequence of the polypeptide is shown as SEQ ID NO.1, and the amino acid sequence of the coding protein is shown as SEQ ID NO. 2. Specific primersTuRLK1-F andTuRLK1the nucleotide sequence of R is as follows:

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

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

as shown in FIG. 1, the TuRLK1 receptor protein kinase of the type PI428309 of the Ula-wheat and the TRILR3_ 02522 receptor protein kinase of the type G1812 of the Ula-wheat have deletions of different amino acid sequences in the LRR (Leucine-Rich Repeat Region) region.

Example 2TuRLK1Regulation in urapidoti wheat PI428309YrU1Mediated stripe rust resistance

cDNA of leaves of urapidotia wheat PI428309 is used as a template, and a primer gamma-TuRLK1F and gamma-)TuRLK1And (3) amplifying R to obtain a PCR product. The method comprises the steps of (1) carrying out enzyme digestion on a Barley stripe mosaic virus RNA gamma (BSMV) plant virus vector by utilizing restriction enzyme Nhe I to obtain a linearization plasmid carrying enzyme digestion sites, and constructing a PCR product into a silencing vector BSMV by utilizing a homologous recombination kit Vazyme ClonExpress: II One Step Cloning Kit C112 to obtain the silencing vector BSMV: TuRLK1. Viral vector BSMV with GFP fragment ligated: GFPfor comparison, the method provided by [ Holzberg S, brosio P, gross C, pogue GP. 2002. Barley stripe mosaic virus-induced gene silencing in a monocot Plant, plant J30:315-327 ] is used to infect one-leaf one-heart-stage Ula drawing wheat PI428309, after virus infection, the fourth leaf is inoculated with Rumex striolatus CY33 ex vivo and testedTuRLK1Expression level of (2); the uracratia material G1812 infected with the rust CY33 is used as a negative control, and the uracratia wheat PI428309 (CK) which is not infected by viruses is used as a positive control. Primer gamma-TuRLK1F and gamma-)TuRLK1The nucleotide sequence of R is as follows:

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

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

the results found (fig. 2), relative to the control BSMV:GFP，BSMV: TuRLK1group ofTuRLK1Down-regulation of expression levels and silencingTuRLK1Ula drawing wheat PI428309 pair capable of causing stripe rust resistancePstCY33 is susceptible to disease. This experiment strongly demonstratesTuRLK1Involved in regulating and controlling urapidoti wheat PI428309YrU1Mediated stripe rust resistance.

Sampling at different time periods after inoculating the rust bacteria, staining a fungus structure by using wheat germ lectin (WGA), and counting indexes such as germination conditions, hypha length, hypha infection area and the like of the rust spores of 2 dpi, 3 dpi and 5 dpi. As a result, it was found (FIG. 3) that BSMV was on day 2 after inoculation:TuRLK1HB (hyphal branching) number of groups compared to control BSMV:GFPabout 2.26 and 1.90, respectively; BSMV:TuRLK1HMC (haustorium master cells) number of groups compared to control BMSV:GFPabout 2.08 and 1.82, respectively; BSMV:TuRLK1the number of H (aspirators) of the group is greater than the control BSMV:GFPabout 1.24 and 1.04, respectively; on day 3 post inoculation, BSMV:TuRLK1hypha germination length of the group was longer than that of the control group BSMV:GFPthe lengths were about 113.92 μm and 87.05 μm, respectively, and were found to have very significant differences by t-test; on day 5 post inoculation, BSMV:TuRLK1hyphal invasion area of the group compared to control BSMV:GFPobviously increase to about 17242.12 μm respectively ² And 11325.74 μm ² With very significant differences. These results indicate that whenTuRLK1Down-regulation, beneficial to the development of the rustTuRLK1Plays an important role in the resistance of the Ula wheat stripe rust CY 33.

Example 3 common wheat overexpressionTuRLK1Can enhance powdery mildew resistance

Reference [ Shen Q-H, saijo Y, mauch S, biskup C, bieri S, keller B, seki H, Ü lker B, somsich IE, schulze-Lefert P.2007 Nu ]clear Activity of MLA Immune Receptors Links Isolate-Specific and Basal Disease-Resistance responses.science 315:1098-1103 by single cell transient expression experimentsTuRLK1And verifying powdery mildew resistance function. On the leaf of common wheat field for powdery mildew infection, gene gun is used to mediate single cell transient expressionTuRLK1(to be in gateway systemTuRLK1The coding region of (2) was ligated into the intermediate vector pDONR207 and transferred into the final vector pUBI-GW, and amplifiedTuRLK1The primers used in the coding region of (2) areTuRLK1attB-F andTuRLK1attB-R), expression 4 h post inoculationBgtE09 GUS staining after 48, h, and aspirator index were counted. Primer(s)TuRLK1attB-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 overexpressionTuRLK1Compared with the control treatment (over-expression of PGY protein), the powdery mildew absorber index on the infected wheat field leaf can be remarkably reduced, and the powdery mildew resistance is improved.

Example 4 heterologous overexpressionTuRLK1Enhancing powdery mildew resistance of Arabidopsis thaliana

Constructing a receptor-like protein kinase gene driven by a constitutive CaMV 35S promoter by taking pEARLEY201 plasmid as a basic vectorTuRLK1Plant expression vector pEARLEY201-TuRLK1The method comprises the steps of carrying out a first treatment on the surface of the The agrobacterium GV3101 strain is transferred by freeze thawing process and the arabidopsis thaliana is genetically transformed by inflorescence dip-dyeing process. Screening positive plants by using herbicide Basta, and detecting positive plantsTuRLK1Is a target expression level. Arabidopsis powdery mildew susceptibility mutantpad4Is a negative control.

To clarify the heterologous overexpression of receptor-like protein kinase genesTuRLK1Effect on powdery mildew resistance function of arabidopsis, inoculation powdery mildew experiments were performed on transgenic arabidopsis at 4 weeks of age. As a result, it was found (FIG. 5) that after 7 days of inoculation, the leaf surfaces of wild type Arabidopsis thaliana were densely packedConidia, and no cell death, butTuRLK1Cell death occurs at powdery mildew infection positions of leaves of the transgenic strain; trypan blue staining observations also found that,TuRLK1powdery mildew conidiophore and hypha on transgenic plant leaves are obviously reduced, and a plurality of cell death can be observed; quantitative inoculation of powdery mildew and further analysis also found that T at 4 weeks of age ₂ On the generation positive transgenic plants, the number of conidiophores produced by germination of single powdery mildew spores (50 single spore colonies per material) was significantly less than that of wild-type arabidopsis (fig. 5). From the above data, it can be seen that when powdery mildew infects, it is heterologously expressedTuRLK1Can induce the leaf of Arabidopsis to generate cell death, thereby inhibiting invasion and growth of pathogenic bacteria and enhancing powdery mildew resistance of Arabidopsis.

The foregoing has been described in some detail by way of illustration of the general principles and specific embodiments of the invention. Certain modifications and derivatives of this invention will be readily apparent to those skilled in the art, and it is therefore intended to be within the scope of this invention as claimed herein without departing from the essential characteristics thereof.

SEQUENCE LISTING

<110> Fujian university of agriculture and forestry

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ggggaccact ttgtacaaga aagctgggtc ctaggagatg accgcctccg cccac 55

Claims

1. Ula diagram wheat receptor protein kinase geneTuRLK1The method is characterized in that: ula drawing wheat receptor protein kinase geneTuRLK1The nucleotide sequence of (2) is shown as SEQ ID NO. 1.

2. An uralensis wheat receptor protein kinase gene according to claim 1TuRLK1The encoded protein is characterized in that: the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. A method for producing a protein kinase gene comprising the uralensis wheat receptor of claim 1TuRLK1Is a recombinant vector of (a).

4. Overexpression of the uracratia wheat receptor protein kinase gene according to claim 1TuRLK1The application in constructing powdery mildew resistant wheat varieties.

5. Overexpression of the uracratia wheat receptor protein kinase gene according to claim 1TuRLK1The application in construction of powdery mildew resistant Arabidopsis materials.