Application of NtRLK2 gene to resistance of tobacco to bacterial wilt

A technology against bacterial wilt and tobacco, applied in application, genetic engineering, plant genetic improvement, etc., to achieve the effect of improving resistance and disease resistance

Inactive Publication Date: 2017-06-20
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, the gene was cloned from tobacco high bacterial wilt resistance variety line 3 and used to construct overexpression and RNAi expression vectors, and transformed into Cuibi No.1 tobacco variety susceptible to bacterial wilt. It is preliminarily predicted that this gene may be an interaction gene of endogenous disease resistance genes, which inhibits the expression of disease resistance genes

Method used

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  • Application of NtRLK2 gene to resistance of tobacco to bacterial wilt
  • Application of NtRLK2 gene to resistance of tobacco to bacterial wilt
  • Application of NtRLK2 gene to resistance of tobacco to bacterial wilt

Examples

Experimental program
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Effect test

Embodiment 1

[0019] [Example 1] RACE acquisition NtRLK2 Gene 3' Unknown Sequence and 5' Unknown Sequence

[0020] Candidate gene fragments were obtained from the results of tobacco 454 sequencing. According to the transcription of tobacco abiotic and biotic stress 454 sequencing transcripts, the peanut expression profile gene chip was used to screen the genes before and after induction of R. solanacearum by chip hybridization before and after inoculation of resistant varieties To obtain candidate gene fragments through differential expression, design a pair of gene primers 9-F (5′-AGCAGACTATCTAAGGGAAG-3′) and 9-R (5′-CTTACTCCATCTGCTTTTCTGATCCAC-3′), and design a pair of primers RACE-F ( 5′-AAGCAGTGGTATCAACGCAGAGTGGCCAT-3′) and RACE-R (5′-ATTCTAGAGGCCGAGGCGGCCGACATGd(T)30N-1N-3′ (N=A, G, C, or T; N-1=A, G, or C)) . Use P9-R primers and RACE-F primers and 9-F primers and RACE-R primers to perform 5' and 3'-RACE reactions respectively. The 5'-RACE reaction conditions are 94°C 5min→(94°C 30s...

Embodiment 2

[0024] [Example 2] NtRLK2 Construction and verification of overexpression vector

[0025] Amplified from a plasmid with a complete reading frame including the stop codon by the pair of primers NtRLK2-OE-F (5’-CGGCTCTAGAACCATGAAGCAAATTCTGATGGACAG-3’) and NtRLK2-OE-R (5’-CAACGGTACCCATAACATTCTCCAATGGTTG-3’) NtRLK2 Gene

[0026] cDNA open reading frame, with XbaI and KpnI restriction sites at the 5' end and 3' end respectively, for the pSC1301 driven by 2×CaMV 35S promoter constructed in our laboratory and the amplified NtRLK2 The target fragment was double digested with XbaI and KpnI at the same time, the target fragment was recovered, ligated with T4 ligase at 16°C overnight, and transformed into E. coli DH5α strain to construct pSC1301- NtRLK2 overexpression vector ( image 3 A).

[0027] Two pairs of primers NtRLK2-RNAi-SpeⅠ-F (5'-CAACACTAGTGAGCTTTTGTAATATCTGCA-3') and NtRLK2-RNAi-KpnⅠ-R (5'-AATTGGTACCGTCTGTTCCAAGAGCTTCC-3') and NtRLK2-RNAi-XbaⅠ-F were designed in its non-...

Embodiment 3

[0029] [Example 3] Genetic transformation of tobacco and PCR identification of transgenic tobacco

[0030] The leaf disk method mediated by Agrobacterium tumefaciens was used to transform tobacco, and the plasmids pSC1301- NtRLK2 The overexpression vector and the Agrobacterium pSC1300-347-NtRLK2 RNAi interference vector were transformed into tobacco variety Cuibi No. 1 (CB-1) by the leaf disk method, and 15 mg / L Hyg was used to screen the growth of leaf buds and roots. During the cultivation process, 500 mg / L (Cef) to inhibit the growth of Agrobacterium, the culture condition temperature is 25°C±2°C, and the light is 14h day / 10h night every day. Co-cultivation medium: MS medium + 0.1mg / L NAA + 1mg / L 6-BA, induction screening medium: MS medium + 0.1mg / L NAA + 1mg / L 6-BA + 15mg / L Hyg + 500mg / L Cef, rooting medium: MS medium + 15mg / L Hyg + 500mg / L Cef.

[0031] For the PCR detection of overexpressed transgenic tobacco, according to the 35S promoter and NtRLK2 Gene sequence de...

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Abstract

The invention relates to a sequence and functional application of an LRR (Leucine-Rich Repeat) acceptor protein kinase gene NtRLK2 related to the resistance of tobacco to bacterial wilt, and belongs to the technical field of molecular biology. According to the sequence and the functional application, through the analysis of a gene chip, a partial segment of one LRR acceptor protein kinase gene NtRLK2 of which the expression is up-regulated by 2 times when being induced by ralstonia solanacearum is obtained; a full-length cDNA (complementary Deoxyribonucleic Acid) sequence of the LRR acceptor protein kinase gene NtRLK2 is obtained through an RACE (Rapid Amplification of cDNA end) technique; the gene is cloned from a high-bacterial-wilt-resistance variety line 3 of the tobacco, is used for constructing over-expression and RNAi (Ribonucleic Acid interference) expression vectors, and is transferred into a bacterial wilt infected variety Cuibi 1# in the tobacco; indicated by inoculation identification, the RANi of the gene obviously enhances the disease resistance of a tobacco plant to the bacterial wilt; preliminarily predicted, the gene is possibly an interacting gene of an endogenous disease-resistant gene; the expression of the disease-resistant gene is inhibited. According to the application, a foundation is about to be laid for the bacterial-wilt disease-resistant genetic breeding of the tobacco.

Description

technical field [0001] The present invention relates to NtRLK2 The application of genes in tobacco bacterial wilt resistance belongs to the technical field of plant genetic engineering. Background technique [0002] Plants go through a complex developmental process from embryogenesis to complete plants, the entire life cycle. This process is jointly regulated by its own genetic material and the environment. In essence, it is a process of how to coordinate the living environment and its own genetic information, so that the regulatory genes are expressed in an orderly manner in time and space, and complete the process of life activities. This requires the existence of receptors for receiving internal and external signal molecules in individual plants, and protein kinases play an important role in a series of processes of this information transduction. Eukaryotic protein kinases catalyze the amino acid side chains of proteins by transferring the phosphate group of ATP, which...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/82A01H5/00
CPCC12N9/12C12N15/8218C12N15/8281C12N2830/34
Inventor 庄伟建张冲陈顺辉庄春红陈华蔡铁城邓烨巫升鑫
Owner FUJIAN AGRI & FORESTRY UNIV
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