Double-stranded RNA molecule for targeted silence of phytophthora capsici cellulose synthase 3 and application of double-stranded RNA molecule

A technology of cellulose synthase and Phytophthora capsici, which is applied in the field of agricultural biology, can solve problems such as undiscovered plant diseases, and achieve the effects of delaying the emergence of drug resistance, inhibiting infection, and reducing the amount of use

Pending Publication Date: 2022-08-05
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there is currently no relevant literature on the control of plant diseases caused by Phytophthora capsici based on RNA silencing technology

Method used

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  • Double-stranded RNA molecule for targeted silence of phytophthora capsici cellulose synthase 3 and application of double-stranded RNA molecule
  • Double-stranded RNA molecule for targeted silence of phytophthora capsici cellulose synthase 3 and application of double-stranded RNA molecule
  • Double-stranded RNA molecule for targeted silence of phytophthora capsici cellulose synthase 3 and application of double-stranded RNA molecule

Examples

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Effect test

Embodiment 1

[0031] Example 1 Source of double-stranded RNA molecule for inhibiting the expression of Phytophthora capsicum cellulose synthase 3 gene involved in the present invention

[0032] (1) The sequence of the double-stranded RNA molecule of the present invention is derived from the cDNA of PcCesA3. The cDNA sequence of PcCesA3 was derived from the gene numbered JX905357 in the database (GenBank) of Phytophthora capsici.

[0033] (2) A total of 555 base sequences numbered JX905357, Nos. 3005-3559 in the cDNA sequence database (GenBank) of PcCesA3 were selected as the sequence of the double-stranded RNA molecule involved in the present invention. Its sense strand sequence is shown in sequence 1 in the sequence listing, and its antisense strand is shown in sequence 2 in the sequence listing.

[0034](3) The above sequences were compared with the genome sequences (NCBI) of common hosts of Phytophthora capsicum such as tobacco, tomato, and pepper, respectively, to detect the possibilit...

Embodiment 2

[0035] Example 2 Preparation of double-stranded RNA molecule of the present invention

[0036] (1) Double-stranded RNA expression plasmid L4440 contains two bidirectional T7 promoters, which can be transcribed in RNaseⅢ-deficient Escherichia coli to generate double-stranded RNA. The target fragment was amplified by PCR, and the amplification primers were shown in Table 1. Using the cDNA of Phytophthora capsici standard strain LT1534 (doi: 10.1094 / MPMI-02-12-0028-R) as a template, the amplified fragment was inserted between the two T7 promoters of the L4440 plasmid by enzymatic ligation (Between the recognition sites of Sac II and Xba I enzymes), after verification by PCR and sequencing, a plasmid capable of expressing double-stranded RNA molecules is obtained.

[0037] Table 1 Sequences of primers for constructing double-stranded RNA molecules

[0038]

[0039] (2) Transform the plasmid capable of expressing double-stranded RNA molecules obtained in step (1) into RNaseIII...

Embodiment 3

[0046] Example 3. Evaluation of disease resistance of plants to which the double-stranded RNA molecule of the present invention is applied to Phytophthora capsici

[0047] In this patent, a. double-stranded RNA molecules are directly sprayed on the surface of the host plant (in vitro leaves of Nicotiana benthamiana and pepper) for 1 day and then inoculated with Phytophthora capsicum zoospores, and the diameter of leaf lesions is observed and measured after 3 days. b. First irrigate the root plants with double-stranded RNA molecules, and then inoculate the pepper seedlings with zoospores in a simulated field environment to determine the disease index of the pepper seedlings. According to the above two evaluation methods, the effect of double-stranded RNA on the growth, development and pathogenicity of Phytophthora capsicum was clarified.

[0048] Phytophthora capsicum BYA5 (isolated by the Laboratory of Fungicide Pharmacology and Pathogen Resistance of China Agricultural Univer...

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Abstract

The invention discloses a double-stranded RNA (Ribonucleic Acid) molecule capable of efficiently inhibiting the expression of a phytophthora capsici cellulose synthase 3 gene, and belongs to the technical field of agricultural biology. The double-stranded RNA molecule disclosed by the invention consists of nucleic acid sequences as shown in SEQ ID No. 1 and SEQ ID No. 2. The invention also discloses application of the double-stranded RNA molecule in preventing and treating plant diseases caused by phytophthora capsici. The double-stranded RNA molecule disclosed by the invention can be used for inhibiting infection of phytophthora capsici by directly treating the phytophthora capsici or spraying the phytophthora capsici on plants. Meanwhile, the double-stranded RNA provided by the invention has strong specificity on essential genes for growth and development of the phytophthora capsici, does not have the problem of drug resistance, and can provide an effective way for preventing and treating related diseases caused by the phytophthora capsici when being used for preparing biopesticides.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a double-stranded RNA molecule that can inhibit the expression of Phytophthora capsici cellulose synthase 3 gene, and the application of the molecule in preventing and treating plant diseases caused by Phytophthora capsici. Background technique [0002] Phytophthora capsici is an important phytopathogenic oomycete that is distributed all over the world and can infect more than 70 species of Solanaceae, legumes and most cucurbits, causing damping, wilting and root, Stem and fruit rot, from seedling stage to fruit stage, can cause serious economic losses. The pepper blight caused by Phytophthora capsicum is a devastating disease that can be spread by rain, soil and air currents, causing symptoms such as leaf withering, fruit rot, and even the death of the whole plant, which has a great impact on crop yield and quality. It can lead to a reduction of more than 50% in produc...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/82
CPCC12N15/1137C12N9/1059C12N15/8282
Inventor 刘西莉王治文高翔李瑜钟珊张博瑞张思聪李腾蛟
Owner CHINA AGRI UNIV
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