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DsRNA for inhibiting rhizopus stolonifer and application of dsRNA

A technology of Rhizopus stolonifera and an inhibitor is applied to the dsRNA inhibiting Rhizopus stolonifera and its application field, which can solve problems such as harming human health and producing resistance of microorganisms, so as to alleviate symptoms of soft rot and prevent and control peach soft rot. effect of disease

Pending Publication Date: 2021-10-01
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, acidic calcium sulfate, chlorine dioxide, potassium sorbate and other chemical fungicides are commonly used at home and abroad to inhibit the growth of Rhizopus stoloniferus, but long-term use of chemical fungicides will not only make microorganisms resistant, but also endanger human health.

Method used

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  • DsRNA for inhibiting rhizopus stolonifer and application of dsRNA
  • DsRNA for inhibiting rhizopus stolonifer and application of dsRNA
  • DsRNA for inhibiting rhizopus stolonifer and application of dsRNA

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Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1: Obtaining of full-length cDNA and target fragments of Rhizopus stolonifer dcl1 and p450 genes

[0021] The target for preventing and controlling Rhizopus stolonifer consists of the dcl1 gene target sequence and the p450 gene target sequence shown in SEQ ID NO.2. Based on the genome database of Rhizopus stoloniferus, the full-length cDNA sequences of the dcl1 and p450 genes of Rhizopus stoloniferus were searched by biological information method, and the non-conserved region sequences were obtained by the software Snap Gene Viewer , which is the target sequence. According to the target sequence, Primer Premier software was used to design upstream and downstream primers for amplifying the target sequence.

[0022] The primers for amplifying the target sequence are:

[0023] dcl1-F: AGGACCTTGGAGTTGT (SEQ ID NO. 5)

[0024] dcl1-R: AAACATTGGGATTACG (SEQ ID NO. 6)

[0025] p450-F: TCTTACTTGTTGCCAAAC (SEQ ID NO. 7)

[0026] p450-R: GTCGCATAAAGGGAGG (SEQ ID N...

Embodiment 2

[0042] Example 2: Obtaining dcl1 and p450 target sequence dsRNA

[0043] First, the T7 promoter was introduced into the 5' and 3' ends of the target sequence by PCR. After purification, the DNA fragment with the T7 promoter at both ends was used as a template to synthesize dsRNA in vitro using the In vitro Transcription T7 Kit. The specific steps are as follows:

[0044] (1) Configure the following reaction solution

[0045] components Dosage 10×Transcription Buffer 2μL ATP solution 2μL GTP solution 2μL CTP solution 2μL UTP solution 2μL RNase Inhibitor 0.5μL T7 RNA Polymerase 2μL PCR product 20ng~1μg RNase free dH 2 o

Add to 20μL Total 20 μL

[0046] (2) Mix the above solutions evenly and centrifuge slightly, collect the transcription reaction solution at the bottom of the reaction tube, and react at 42°C for 1-2 hours.

[0047] (3) DNase I treatment: after the transcription reaction, 2 ...

Embodiment 3

[0057] Embodiment 3: the application of dcl1 and p450 gene dsRNA in the prevention and treatment of stoloniferous rhizopus

[0058] For dsRNA transcribed in vitro, adjust the concentration to 20ng / μL (use RNase-free water) before use.

[0059] A number of fresh peaches were purchased from Nanjing Zhongcai Vegetable Wholesale Market, and peaches of uniform size and maturity were selected, cleaned, disinfected, and dried in a fume hood.

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Abstract

The invention belongs to the technical field of biology, and particularly relates to dsRNA for inhibiting rhizopus stolonifer and application of the dsRNA. The sequence of the dsRNA for inhibiting the rhizopus stolonifer is shown as SEQ ID NO.1. The invention discloses application of the dsRNA in preparation of a rhizopus stolonifer inhibitor. Rhizopus stolonifer dcl1 and p450 genes are provided for the first time, non-conserved region sequences of the rhizopus stolonifer dcl1 and p450 genes are selected as target sequences, upstream and downstream primers are designed according to the target sequences and used for amplifying the target sequences, T7 sequences are added to the two ends of the target sequences, and dsRNA is synthesized. A plate bacteriostasis experiment shows that the dsRNA can remarkably inhibit extension and diffusion of rhizopus stolonifer hyphae. It is found that the soft rot symptom of peaches added with the dsRNA is obviously relieved by spraying the dsRNA outside the peach bodies, and therefore the purpose of preventing and treating the soft rot of the peaches is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a dsRNA for inhibiting rhizopus stoloniferus and application thereof. Background technique [0002] Rhizopus stolonifer (Rhizopus stolonifer) belongs to Mucorales, Mucorales, and Rhizopus stolonifer. It is a strong parasitic pathogen. Grow and reproduce. Rhizopus stolonifera can infect peaches, apricots, pears, kiwis, melons, grapes, enoki mushrooms, sweet potatoes and other fruits and vegetables, causing soft rot and causing huge losses to agricultural production. At present, acidic calcium sulfate, chlorine dioxide, potassium sorbate and other chemical fungicides are commonly used to inhibit the growth of Rhizopus stoloniferus at home and abroad, but long-term use of chemical fungicides will not only make microorganisms resistant, but also endanger human health. Therefore, it is urgent to find an efficient, safe and environmentally friendly method for the prevention a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/31A01N57/16A01P3/00
CPCC12N15/113C07K14/37A01N57/16C12N2310/14Y02A50/30
Inventor 张映曈周宏胜凌军李鹏霞赵欢欢
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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