Cloning of gene against meloidogyne of capsicum and application thereof

A technology of anti-root-knot nematode and pepper genome, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems that did not mention tomato, the application method and application effect of the gene, and solve the problem of lack of germplasm resources, The effect of saving production cost and shortening the breeding period

Inactive Publication Date: 2007-04-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it is mentioned that it is a Solanaceae plant in the patent of the previous application, it does not mention tomato and the application method and application effect of the gene in tomato in the instructions and examples; and the present invention clones the anti- root-knot nematode gene, and then use it in the breeding of tomato root-knot nematode-resistant transgenic plants, with detailed preparation methods and application effect instructions

Method used

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  • Cloning of gene against meloidogyne of capsicum and application thereof
  • Cloning of gene against meloidogyne of capsicum and application thereof
  • Cloning of gene against meloidogyne of capsicum and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: the isolated clone of Me gene of the present invention

[0047] The cloned gene of the present invention comes from the capsicum genome, according to the four anti-nematode genes that have been cloned in the plant (referring to the above-mentioned reference Hsl pro-1 ①, Mi②, Gpa2③, Hero④) nucleotide binding site (Nucleotide binding site, NBS) and leucine-rich repeat region (Leucine-rich repeat region, LRR) conservative sequence design a pair of degenerate primers (as described below ), using the resistant pepper genomic DNA as a template, PCR amplification, the reaction system is 25μL, containing 1×PCR buffer, 1.5mmol / LMgCl 2 , 0.2mmol / L dNTPs, 0.2mmol / L forward and reverse primers, 1U Taq DNA polymerase, 100ng template. Reaction cycle parameters: 94°C pre-denaturation for 5min, 94°C for 30s, 50°C for 1min, 72°C for 90s, 5 cycles, 94°C for 30s, 55°C for 1min, 72°C for 90s, 25 cycles, 72°C for 10min, 4°C storage . The PCR product was recovered, connected...

Embodiment 2

[0059] Embodiment 2, plant transformation vector construction

[0060] The plasmid pDAMe was digested with ApaI and NruI, and then filled with T4 DNA Polymerase to recover the large fragment; at the same time, the plasmid pBI121 was digested with BamHI and SacI to remove the GUS gene fragment, filled with T4 DNA Polymerase, and then acidic phospholipid Dephosphorylate the enzyme CIAP, recover large fragments after gel electrophoresis, ligate with the previously recovered product under the action of T4 DNA ligase, transform the ligated product into E. coli strain DH5α, and place it on an LB solid plate containing 50 mg / L kanamycin Positive clones were screened, plasmids were extracted for enzyme digestion and PCR identification, and a recombinant clone containing the inserted target fragment was obtained, which was named pBAMe. Application of electric shock method (see J. Sambrook et al., "Molecular Cloning Experiment Guide", third edition, translated by Jin Dongyan et al., Sci...

Embodiment 3

[0061] Embodiment 3, plant genetic transformation

[0062] The plasmid pBAMe was introduced into Agrobacterium LBA4404 by electric shock method, and the tomato variety "Zhongshu No. 5" (purchased from a commercial variety) with stable traits and sensitivity to root-knot nematode was transformed. Molecular identification, insect resistance screening and self-purification , to obtain homozygous transgenic plants or strains.

[0063] The genetic transformation steps of tomato mediated by Agrobacterium tumefaciens are as follows:

[0064] 1. Sterile vaccine and tobacco cell culture

[0065] Aseptic seedling cultivation: Disinfect the seeds of the tomato variety "Zhongshu No. 5" with 70% alcohol for 1 min, then sterilize in 20% sodium hypochlorite (containing 6% available chlorine) for 15 min, rinse with sterilized distilled water for 3 times, and inoculate the seeds in 1 / 2 MS basic medium (see Murashige T. and F. Skoog. Physiol. Plant, 1962, 15: 473-497), in the dark at 25±2°C u...

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Abstract

The invention belongs to the field of plant genetic engineering technologies. The invention specifically involves the separation and cloning of a DNA fragment. The said DNA fragment gives the plants the tolerant capacity against diseases caused by edlworm. Inserting the DNA fragment into proper vector to obtain plant expressing vector (conservation number: CCTCC-M205110), introducing the plant expressing vector into plant through Agrobacterium-mediated method and the transgenic plants can generate resistance against edlworm. As one of the implementation of cases, nematode resistant transgenic tomatoes have been obtained.

Description

technical field [0001] This patent belongs to the field of plant genetic engineering. It specifically involves isolating and cloning the root-knot nematode resistance gene from the resistant pepper genome, and then introducing the gene into plant materials without root-knot nematode resistance, such as tomato sensitive to root-knot nematode, to obtain transgenic root-knot nematode-resistant Nematode tomato plants. Background technique [0002] Plant root-knot nematode (Meloidogyne spp.) disease is a worldwide disease that seriously threatens crop production. Root-knot nematode has a wide range of hosts (up to more than 2000 kinds of plants) and various transmission routes. It is a soil-borne disease source that is extremely difficult to control. There are more than 80 kinds of root-knot nematodes that have been found in the world (Karssen et al., 1999), and 25 species have been identified in my country, among which the most widely distributed and most harmful ones are M. N...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82
CPCY02A40/146
Inventor 叶志彪陈儒钢李汉霞欧阳波卢永恩张俊红
Owner HUAZHONG AGRI UNIV
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