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Method for doubly preventing tomato root-knot nematode by combining efficient RNA (Ribonucleic Acid) with Bt gene

A technology of RNA interference and gene, applied in the field of plant disease and insect pest control, can solve the problem of limited control effect and achieve the effect of effective nematode control strategy and reliable nematode control strategy

Inactive Publication Date: 2013-05-08
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the transformation of a single gene has limited efficacy

Method used

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  • Method for doubly preventing tomato root-knot nematode by combining efficient RNA (Ribonucleic Acid) with Bt gene
  • Method for doubly preventing tomato root-knot nematode by combining efficient RNA (Ribonucleic Acid) with Bt gene
  • Method for doubly preventing tomato root-knot nematode by combining efficient RNA (Ribonucleic Acid) with Bt gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Vector pCAGC1341-TGPM01 RNAi construction process

[0058] The RNA interference-inducing vector pFGC5941 (gifted by the University of Leeds, UK, is a commonly used vector and can be purchased in the market) has a herbicide resistance (BAR) gene, and the restriction site is repeated with the insert, so it is necessary to construct a hygromycet-containing Insert the RNA interference inducing vector of pFGC5941 (3577bp) into the cloning vector pCAMBIA1300 with hygromycin selection marker (preserved by the Laboratory of Vegetable and Flower Research Institute, Chinese Academy of Agricultural Sciences, and can be distributed to the public)

[0059] Digested pFGC5941 and pCAMBIA1300 with EcoRI / PstI respectively, digested overnight at 37°C, and recovered the 8.9kb fragment after electrophoresis of the digested product of pCAMBIA1300, and recovered the 3.5kb fragment of the digested product of pFGC5941 after electrophoresis, and used T4 DNA Ligase ligation, incubated...

Embodiment 2

[0068] Example 2: Construction of a plant expression vector-pBI121-06 expressing Bt-Cry6A

[0069] According to the preference of plants for nucleotide codons, Bt-Cry6A was artificially synthesized, and its nucleotide sequence is shown in Seq ID No.4.

[0070] Construction of vector pBI121-06

[0071] Using the conventional vector pBI-121 as the backbone, use restriction endonucleases XbaⅠ and BamHI to double-enzyme digest the synthesized Bt-Cry6A gene and the backbone vector, respectively, recover the target fragment, connect, identify and transform to obtain a plant expression vector pBI121-06( Figure 4 ).

Embodiment 3

[0072] Example 3: Obtaining of tomato plants transfected with pCAGC1341-TGPM01RNAi vector

[0073] In this experiment, mediated by Agrobacterium tumefaciens, the pCAGC1341-TGPM01 RNAi vector loaded with the MiMPK1 gene fragment was transformed into tomato. A method for obtaining transgenic plants by optimizing transformation conditions.

[0074] 2.1 Cultivation of Agrobacterium tumefaciens

[0075] 1. Streak inoculation of Agrobacterium tumefaciens (EHA105) on LB medium and culture at 28°C for 2-3 days;

[0076] 2. Pick a single colony and inoculate it in the basic medium, shake and culture at 200 rpm for 24 hours at 28°C;

[0077] 3. Collect the bacteria in a centrifuge tube, add liquid induction medium, adjust the OD600 to about 0.20, shake and culture at 28°C and 200rpm for 6 hours, and stop when the OD600 rises to about 0.4.

[0078] 2.2 Agrobacterium competent production:

[0079] (1) Pick a single colony of Agrobacterium EHA105, inoculate it in 20ml liquid LB medi...

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Abstract

The invention discloses a method for doubly preventing tomato root-knot nematode by combining efficient RNA (Ribonucleic Acid) with Bt gene, and belongs to the field of preventing of plant diseases and pests. The method comprises the following steps of: using transgenic tomato which is transferred with an RNA interference vector pCAGC1341-TGPM01RNAi as a host plant transfer explant; transferring a vector of expression Bt-Cry6A crystal protein to obtain a transgenic tomato variety capable of doubly preventing root-knot nematode. The invention further provides an optimization method for trans-gene. The gene synthesized in the invention is obtained by optimizing codon. The optimized artificial gene is coded with Bt-Cry6A crystal protein, and the tomato variety which is transferred with the gene has a good root-knot nematode preventing effect compared with the tomato variety which is transferred with the single gene.

Description

technical field [0001] The invention belongs to the technical field of plant disease and insect pest control. The high-efficiency RNA interference gene and the nematode Bt gene are polymerized and transformed into tomatoes, and the dual effects jointly prevent and control root-knot nematodes. Background technique [0002] Root-knot nematode disease is an important biological disaster that seriously harms various economic crops such as vegetables, cotton, peanuts, and tobacco, and food crops such as rice and corn in China. In recent years, the problem has become more prominent after the adjustment of agricultural planting structure and layout. At present, the control of crop nematode diseases mainly depends on nematicides. Due to the poor control effect of nematicides, agricultural production has been reduced, and unreasonable use has brought product pollution and ecological environment damage. Therefore, the serious harm of crop nematode diseases directly threatens the food ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/32A01H4/00A01H5/00
Inventor 陈国华王殿东田雪亮王运生茆振川杨宇红谢丙炎
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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