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ESCRT-III core subunit Snf7 double-stranded RNA and preparation method and application thereof

An ESCRT-III, the core technology, applied in the field of double-stranded RNA, the preparation of the dsRNA, can solve the problems of restricting the use of insect-specific gene insecticides, limited insect lethal efficiency, and low safety, so as to inhibit normal development and reproduction , significant effect of prevention and control

Active Publication Date: 2018-10-26
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main advantage of these insecticides is that they are broad-spectrum insecticides for most insects, but their relative safety is not high, and their lethal efficiency to insects is limited, which greatly limits the use of insect-specific gene insecticides. use

Method used

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  • ESCRT-III core subunit Snf7 double-stranded RNA and preparation method and application thereof
  • ESCRT-III core subunit Snf7 double-stranded RNA and preparation method and application thereof
  • ESCRT-III core subunit Snf7 double-stranded RNA and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Prepare dsRNA as follows:

[0048] 1. Select brown planthopper adults for 1 day

[0049] 2. Extraction of total RNA: Take 1 adult worm, grind it in liquid nitrogen, and extract total RNA by TRIzol method.

[0050] 3. Synthesis of the first strand of cDNA: According to the instructions of the PrimeScript RT reagent Kit with gDNA Eraser kit from Takara, 1 μg of total RNA was reverse transcribed into cDNA.

[0051] 4. The preparation process of the N1020727 intermediate fragment is described below.

[0052] (1) Polymerase chain reaction (PCR) reagents (Vazyme Max Super-Fidelity DNAPolymerase) and reaction conditions:

[0053]

[0054] The specific steps of the above polymerase chain reaction are as follows: pre-denature the above mixture at 95°C for 3 minutes, and then enter the following cycle: denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 50 s, a total of 30 cycles, and finally 72°C for 30 cycles. Extend for 7min.

[0055] Whe...

Embodiment 2

[0066] (1) Design specific primers with T7 promoter to synthesize dsRNA of N1020727 in vitro, and use primers of green fluorescent protein gene to synthesize dsGFP as a control.

[0067] dsN1020727-F: 5'-AATGAAGGGAAGAGAGATCGTGCCAAA-3' (SEQ ID NO: 4)

[0068] dsN1020727-R: 5'-TACAGCCTCATCATCCTCAGCAGTCA-3' (SEQ ID NO: 5)

[0069] dsNl020727-T7F:

[0070] 5'-GGATCCTAATACGACTCACTATAGGAATGAAGGGAAGAGAGATCGTGCCAAA-3'

[0071] (SEQ ID NO: 6)

[0072] dsNl020727-T7R:

[0073] 5'-GGATCCTAATACGACTCACTATAGGTACAGCCTCATCATCCTCAGCAGTCA-3' (SEQ ID NO: 7)

[0074] dsGFP-F: 5'-AAGGGCGAGGAGCTGTTCACCG-3' (SEQ ID NO: 8)

[0075] dsGFP-R: 5'-CAGCAGGACCATGTGATCGCGC-3' (SEQ ID NO: 9)

[0076] dsGFP-T7F:

[0077] 5'-GGATCCTAATACGACTCACTATAGGAAGGGCGAGGAGCTGTTCACCG-3' (SEQ ID NO: 10)

[0078] dsGFP-T7R:

[0079] 5'-GGATCCTAATACGACTCACTATAGGCAGCAGGACCATGTGATCGCGC-3' (SEQ ID NO: 11)

[0080] (2) Using the plasmid named pEASY--020727 obtained in Example 1 as a template, use the primers designed i...

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Abstract

The invention provides an ESCRT-III core subunit Snf7 double-stranded RNA. A nucleotide sequence of a positive-sense strand of a transcription template is shown in SEQ ID NO: 1.

Description

technical field [0001] The invention belongs to the field of insect control, and in particular relates to a double-stranded RNA (dsRNA) of the core subunit Snf7 of the sorting complex III core subunit of the transport essential body, and a preparation method and application of the dsRNA. Background technique [0002] ESCRT-Ⅲ, known as the endosomal sorting complexes required for transport-III, is mainly composed of four "core" subunits, Vps20, Snf7 / Vps32, Vps24 and Vps2, and is completed in eukaryotic cells Endosomal membranes invaginate to form key components of multivesicular bodies (MVBs). The main function of ESCRT-Ⅲ is to recruit de-ubiquitin enzymes to promote the degradation of ubiquitin-labeled membrane proteins. In addition to participating in the formation of MVB, ESCRT-III is also related to cell life activities such as internalization, transport, sorting, autophagy and cell division of cell transmembrane proteins, and these life processes are closely related to ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10A01N57/16A01P7/04
CPCA01N57/16C12N15/113C12N2310/14C12Q1/686C12Q2521/107
Inventor 张文庆黎丹
Owner SUN YAT SEN UNIV
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