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DsRNA sequence for regulating and controlling male nilaparvata lugens seminal fluid Selenoprotein F and application of dsRNA sequence

A brown planthopper and sequence technology, applied in the field of molecular biology, can solve the problems of shortening sperm lifespan and sperm motility, and achieve the effects of reducing egg production and egg hatching rate, controlling the number of populations, and reducing the number of offspring populations.

Pending Publication Date: 2021-01-08
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Deficiency of selenoproteins leads to reduced sperm motility and shortened sperm lifespan

Method used

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  • DsRNA sequence for regulating and controlling male nilaparvata lugens seminal fluid Selenoprotein F and application of dsRNA sequence
  • DsRNA sequence for regulating and controlling male nilaparvata lugens seminal fluid Selenoprotein F and application of dsRNA sequence
  • DsRNA sequence for regulating and controlling male nilaparvata lugens seminal fluid Selenoprotein F and application of dsRNA sequence

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021]Example 1 Synthesis of dsRNA

[0022](1) Select the adult male brown planthopper in the insect breeding room, and extract the total RNA of the male brown planthopper using SV total isolation system Kit (Promega, Corporation, Madison, WI, USA);

[0023](2) Use Takara's PrimeScriptTM RT reagent Kit with gDNA Eraser kit to remove genomic DNA, and perform reverse transcription to synthesize cDNA template;

[0024](3) Download the full-length sequence of Selenoprotein F from NCBI (gene accession number: XM022339255), and use the software Primer Premier 5.0 to design dsRNA primers that interfere with Selenoprotein F. The dsRNA template sequence length is 226bp, and the primer sequence is shown in Table 1 (underlined T7 promoter), the designed primers were synthesized by the Shanghai Biotechnology Co., Ltd., and PCR amplified by Takara's Premix TaqTM (94℃, pre-denaturation 5min; 94℃, denaturation 40s; 55℃, annealing 40s; 72℃ Extend for 1 min, a total of 30 cycles of reactions; at 72°C and fin...

Embodiment 2

[0030]Example 2 Microinjection of dsRNA

[0031]Pass the newly emerging male brown planthopper to CO2After anesthesia, the synthetic dsRNA of the present invention is injected from the back of the male brown planthopper chest using a microinjector (100ng / head), and the needle stays for 25-30 seconds to pull out; after injection, it will be collected for 1, 3, 5, and 7 days after emergence Nilaparvata lugens samples were tested for changes in the expression level of the Selenoprotein F gene. The control group was injected with dsRNA synthesized with the green fluorescent protein GFP gene as a template.

Embodiment 3

[0032]Example 3 The effect of silencing Selenoprotein F gene on male brown planthopper on sperm transmission

[0033]Take a few males 2 days after emergence from each treatment, and dissect their internal reproductive system in pre-cooled PBS (pH 7.0). Tear open the abdomen with medical dissecting forceps under a stereo microscope (OLYMPUS CX23, Japan), remove the internal genitalia, remove the fat body and microtrachea attached to the internal genitalia, and pass the complete internal genitalia of the male through the stereo microscope (OLYMPUS CX23, Japan) Observe and take pictures with DS-Fi2 digital camera (Nikon, Tokyo, Japan).

[0034]Take the female adults of the brown planthopper 2 days after mating, dissect out the complete sperm vesicles and take the complete internal genitalia of the males 2 days after the emergence of various treatments, remove the fat body and microtrachea attached to the internal genitalia, and wash them with PBS 3 times, 10 min each time, add 4% paraformald...

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Abstract

The invention discloses a dsRNA sequence for regulating and controlling male nilaparvata lugens seminal fluid Selenoprotein F and application of the dsRNA sequence. The dsRNA sequence is shown as SEQID NO. 1. After the dsRNA is injected to interfere with the male nilaparvata lugens Selenoprotein F gene through an RNAi interference technology, the seminiferous duct of the male nilaparvata lugens is remarkably deformed, the quantity of sperms in seminal vesicles of female nilaparvata lugenss mated with the male nilaparvata lugens is remarkably reduced, the egg laying quantity and egg hatching rate of the nilaparvata lugenss are effectively reduced, the nilaparvata lugens population quantity is controlled, and therefore, function of ecological prevention and control is expected to be fully exerted while pests are inhibited. The sequence can be widely applied to crop breeding, biopesticide research and development and biological control.

Description

Technical field[0001]The present invention relates to the technical field of molecular biology, in particular to the dsRNA sequence of the semen Selenoprotein F of the male brown planthopper and its use.Background technique[0002]Nilaparvata lugensIt belongs to the order Hemiptera and the planthopper family. It is the chief pest on rice in my country and many Asian countries. The brown planthopper is a monophagous pest and can only feed and reproduce offspring on rice and ordinary wild rice. When rice is seriously damaged by brown planthoppers, it will cause large-scale deaths of rice, causing "planthopper fire" phenomenon. Once the brown planthopper breaks out, it will cause serious losses to rice production. At present, the control of brown planthopper is still based on chemical control. However, with the long-term and large-scale use of various chemical agents, brown planthoppers are prone to drug resistance, which further promotes the excessive use of chemical agents, causing ser...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11A01N57/16A01P7/04
CPCC12N15/113A01N57/16C12N2310/14
Inventor 戈林泉江璐周泳凯陈宇张杰钰武庆
Owner YANGZHOU UNIV
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