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Functions of imaginal disk growth factors and application of dsRNA of imaginal disk growth factors

A growth factor, adult technology, applied in the function and the application field of dsRNA, can solve the problem that the function needs to be studied and so on

Active Publication Date: 2020-03-27
山西农业大学植物保护学院(山西省农业科学院植物保护研究所)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insect imaginal disk growth factor (IDGF) is a chitinase-like gene unique to insects. Although it has a conserved domain of chitinase, it has no intrinsic role of chitinase, and its function remains to be studied

Method used

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  • Functions of imaginal disk growth factors and application of dsRNA of imaginal disk growth factors
  • Functions of imaginal disk growth factors and application of dsRNA of imaginal disk growth factors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] imaginal disc growth factor gene AiIDGF acquisition of the sequence

[0013] (1) The imaginal disc growth factor gene of the cutworm AiIDGF Search of the sequence of the cutworm transcriptome database

[0014] Based on the transcriptome database of the cutworm, using bioinformatics methods to identify the imaginal disc growth factor genes of cutworms AiIDGF After searching for the sequence, and after sequence analysis and comparison, a total of 1 imaginal disc growth factor gene of Fly cutworm was obtained AiIDGF the sequence of.

[0015] (2) The imaginal disc growth factor gene of the cutworm AiIDGF verification of the sequence

[0016] Based on the obtained imaginal disc growth factor gene of cutworm AiIDGF Using primer premier5.0 software to design primers to verify the full-length mRNA sequence. The primers used were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The total RNA was extracted from the 6th instar nymphs of cutworm and reverse transcribe...

Embodiment 2

[0018] Synthesis of dsRNA of imaginal disc growth factor gene of cutworm

[0019] (1) Design of dsRNA primers for imaginal disc growth factor gene of cutworm

[0020] Based on the sequence SEQ ID NO: 1 of the imaginal disc growth factor gene of cutworms obtained in this study, it was designed using primerpremier5.0 software. Design dsRNA primers, the sequences of which are SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The primers used were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0021] (2) Synthesis of dsRNA of imaginal disc growth factor gene of cutworm

[0022] The above dsRNA primers were used to synthesize PCR products, which were purified by Wizard® SV Gel and PCR Clean-Up System (Promega) kit, and dsRNA was synthesized by in vitro transcription according to the instructions of T7 RiboMAX™ Express RNAi System (Promega) kit. The concentration of dsRNA was determined using a microplate reader SpectraMax 190 and concentrated to a final concentration of 3 μ...

Embodiment 3

[0024] Application of dsRNA of imago disc growth factor gene of cutworm in pest control

[0025] (1) dsRNA injection of the adult disc growth factor gene of the cutworm

[0026] 5 μL (15 μg) of dsRNA was injected with a 25 μL microsyringe between the seventh and eighth abdominal segments of the 2nd-day-old larvae of the 6th-instar cutworm. Inject dsGFP at the same volume concentration into the control group. After the injection, the young cutworms were raised in a constant temperature biochemical incubator at 27°C, and soil substrate was added 1 day later.

[0027] (2) Detection of gene silencing efficiency of adult disc growth factor of cutworm

[0028] After the dsRNA injection of the 6th instar cutworm, the silencing efficiency was detected 24 hours after the injection, and the whole insect body was used as the object of RNA extraction, and 3 biological replicates were set for each group. The total RNA of each sample was extracted and reverse-transcribed into first-stran...

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Abstract

The invention provides functions of imaginal disk growth factors (IDGF) and an application of dsRNA of the imaginal disk growth factors. The imaginal disk growth factors can assist insects to resist germ invasion. When the imaginal disk growth factors keep silent by the dsRNA, the resistibility of the insects is reduced until the insects die. After the imaginal disk growth factor gene of black cutworms is subjected to cloning and sequencing, the obtained sequence is as shown in SEQID NO:1; on the base of the obtained sequence, a fragment of which the sequence is as shown in SEQID NO:2 is selected and is used for synthesis of double-strand RNA(dsRNA); and after the dsRNA is injected into the body cavities of the insects, the phenomena that the resistibility is reduced, the insects are susceptible to germs and melanism appears are presented in the development process of the insects, and the mortality rate is greatly raised. The imaginal disk growth factors disclosed by the invention provide a new target for pest control.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the function of an insect imaginal disc growth factor (IDGF) and the application of dsRNA. Background technique [0002] Agricultural pests are a threat to my country's food safety production. At present, the long-term use of chemical insecticides for prevention and control has led to the emergence of insect resistance; indirectly, the amount of pesticides has increased, resulting in environmental pollution and threats to human health. A series of problems. With the development of actions such as reducing the use of chemical fertilizers and pesticides in my country, new pest control methods and strategies are urgently needed. [0003] The application of RNA interference (RNAi) in pest control has become popular in recent years and is developing at a high speed. This method has the advantages of specific control, green safety and so on. Studying the function of insect g...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/113A01N57/16A01P7/04
CPCA01N57/16C07K14/43563
Inventor 李大琪张建琴陆俊姣任美凤董晋明李涛
Owner 山西农业大学植物保护学院(山西省农业科学院植物保护研究所)
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