Application of CsLYK gene and encoded protein thereof in improving resistance to citrus canker

A citrus canker and protein-encoding technology is applied to the CsLYK gene and its encoded protein to improve the application field of citrus canker resistance, and achieve the effects of reducing the degree of disease, reducing the area of ​​lesions, and having great application value.

Active Publication Date: 2020-02-21
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research and application of using LYKs to improve the resistance of citrus to citrus bacterial canker

Method used

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  • Application of CsLYK gene and encoded protein thereof in improving resistance to citrus canker
  • Application of CsLYK gene and encoded protein thereof in improving resistance to citrus canker
  • Application of CsLYK gene and encoded protein thereof in improving resistance to citrus canker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Cloning of Citrus CsLYK Gene Coding Sequence

[0044] 1. RNA extraction and cDNA synthesis

[0045] Total RNA was extracted from citrus (Wanjincheng) leaves with an RNA extraction kit (Adelaide, CAT: RN09). The RNA quality was verified by agarose gel electrophoresis, and its concentration was measured with a densitometer. Recombinant DNase I was used to synthesize cDNA (TAKARA), and the cDNA was stored at -20°C for future use.

[0046] 2. PCR amplification of CsLYK gene coding sequence

[0047] Use primer OE-F (SEQ ID No.3) and OE-R (SEQ ID No.4) to obtain CsLYK fragment ( figure 1 ), the length of the fragment is 2054bp (including the enzyme cutting site), the amplified DNA fragment is sequenced and analyzed as the coding sequence of the citrus CsLYK gene, and the software analyzes the corresponding amino acid sequence and structural domain to verify the correctness of the protein. It has two LysM domain, a kinase domain, a signal peptide, and a transmembrane domain...

Embodiment 2

[0053] Construction of overexpression vector and transformation of Agrobacterium

[0054] The vector construction flow chart is as follows image 3 , all restriction endonucleases were purchased from (THERMO) company, and operated according to the instruction.

[0055] The specific operation is as follows: the CsLYK gene fragment and the overexpression vector pLGNe were double-digested with restriction endonucleases EcoRI and KpnI, recovered and ligated overnight, using T4 DNA Ligase kit (TAKARA) for ligation. The ligation product was transformed into Escherichia coli DH5α, and the plasmid was extracted from the positive clone to obtain the overexpression vector pLGNe-LYK of CsLYK. Plasmid extraction kits (Adelaide) were used.

[0056] The constructed overexpression vector was introduced into Agrobacterium tumefaciens EHA105 by electric shock method. Take the frozen EHA105 Agrobacterium competent cells (50 μL) in advance, thaw on ice, add 2 μL of the constructed overexpress...

Embodiment 3

[0058] Genetically Transformed Citrus (Wanjincheng)

[0059] 1. Acquisition of epicotyls from citrus seedlings

[0060] Fresh citrus (Wan Jincheng) fruit is washed, 70% alcohol surface disinfection, the seeds are taken out under aseptic conditions, the seed coat is peeled off, germinated on the seed germination medium, cultivated in the dark at 28°C for 2 weeks, and then in 16h light / Incubate for 1 week under 8h dark conditions. Under sterile conditions, the epicotyls of germinated seedlings were cut into 1 cm stem segments for genetic transformation of Agrobacterium tumefaciens.

[0061] 2. Preparation of Agrobacterium tumefaciens

[0062] The Agrobacterium bacteria solution (containing the CsLYK overexpression vector) used for transfection was added with 30% sterile glycerol and stored in an ultra-low temperature incubator at -80°C. Before transfection, streak culture on LB solid medium containing 50 mg / L kanamycin. Pick a single colony, inoculate in 25ml LB liquid medi...

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Abstract

The invention discloses application of CsLYK gene and an encoded protein thereof in improving resistance to citrus canker. The encoded protein of the CsLYK gene is a citrus LysM-like receptor proteinphosphokinase, and is named CsLYK protein. The nucleotide sequence of the CsLYK gene is a nucleotide sequence shown in SED ID No. 1; the protein sequence of the CsLYK gene is an amino acid sequence shown in SED ID No. 2. The invention also protects a breeding method of citrus resisting bacterial canker, and the method comprises following steps of integrating a citrus LysM-like receptor protein phosphokinase-encoding gene into a citrus through an overexpression vector to effectively improve citrus resistance to bacterial canker. The invention has great application value for breeding of citrus resisting bacterial canker; thereby laying the foundation of genetic breeding of citrus resisting canker, and vigorously promoting the development and application of citrus resisting canker genetic engineering.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the application of a CsLYK gene and its encoded protein in improving resistance to citrus canker. Background technique [0002] Citrus is the largest fruit in southern my country and an important economic crop in the world. However, Citrus bacterial canker (CBC) has seriously hindered the healthy development of the citrus industry (Hu Junhua et al., 2015). Citrus canker is caused by Xanthomonas citri subsp.citri (Xcc), which originated in India, Java and other places (Hu Junhua et al., 2015). The main citrus producing areas such as Fujian, Hunan, and Guangdong in my country are seriously affected by citrus canker. Canker bacterium mainly infects citrus leaves, branches, and fruits, among which seedlings and saplings are more severely damaged (He Xiuling et al., 2007). Diseased trees will have fallen leaves, dead shoots, tree vigor and fruit drop, which seriously affect the yie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/82A01H5/00A01H6/78C12Q1/6895
CPCC12N9/12C12N15/8281C12Q1/6895C12Y207/11C12Q2600/158C12Q2600/13
Inventor 李强祁静静胡安华何永睿陈善春
Owner SOUTHWEST UNIVERSITY
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