Title:
CONSTRUCTION METHOD FOR SYSTEM FOR LARGE VOLUME IN VITRO ACQUISITION OF ERICERUS PELA DSRNA
Document Type and Number:
WIPO Patent Application WO/2021/232536
Kind Code:
A1
Abstract:
Provided is a construction method for a system for large volume in vitro acquisition of ericerus pela dsRNA, the method comprising first extracting a total RNA of Ericerus pela and, after reverse transcription to cDNA, performing PCR amplification, respectively double digesting an amplified product and an L4440 vector plasmid, ligating a double digestion product, transforming and adding an obtained recombinant plasmid into an E. coli competent cell HT115 (DE3) strain, spreading to a solid culture medium and selecting a single colony, and subsequently performing shaker culturing on a single colony liquid until OD600 = 0.38-0.42, adding an IPTG inductor and then concentrating a bacterial liquid, which is used for an RNA interference experiment. By means of single cell cloning of E. coli to have an Ericerus pela target gene, and further performing propagation by means of the innate metabolism and an extensive amount of self-reproduction of said E. coli, a large volume of bacterial liquid containing dsRNA of the Ericerus pela gene is obtained; the dsRNA-containing bacterial liquid is sprayed on Ericerus pela larvae, which enters into the body from the surface of the Ericerus pela, effectively interfering expression of the target gene.
Inventors:
CHEN HANG (CN)
LING XIAOFEI (CN)
LU QIN (CN)
ZHANG JINWEN (CN)
WANG WEIWEI (CN)
LIU PENGFEI (CN)
WANG WEI (CN)
CHEN XIAOMING (CN)
LING XIAOFEI (CN)
LU QIN (CN)
ZHANG JINWEN (CN)
WANG WEIWEI (CN)
LIU PENGFEI (CN)
WANG WEI (CN)
CHEN XIAOMING (CN)
Application Number:
PCT/CN2020/098184
Publication Date:
November 25, 2021
Filing Date:
June 24, 2020
Export Citation:
Assignee:
RES INSTITUTE OF RESOURCE INSECT CHINESE ACADEMY OF FORESTRY (CN)
International Classes:
C12N15/10; A01N57/16; A01P7/04; C12N15/113; C12N15/70
Foreign References:
| CN109929871A | 2019-06-25 | |||
| CN106119258A | 2016-11-16 | |||
| US20050124703A1 | 2005-06-09 |
Other References:
WANG XUE-QING;ZHAO ZUN-LING;SUN TAO;CHEN XIAO-MING;YANG PU: "Construction of RNA Interference Vector of Ericerus pela(Chavannes) ws Gene and Preparation of dsRNA by Prokaryotic Expression", FOREST RESEARCH, vol. 31, no. 4, 15 August 2018 (2018-08-15), pages 70 - 74, XP055870164, ISSN: 1000-1498, DOI: 10.13275/j.cnki.lykxyj.2018.04.010
YANG PU, ZHU JIA-YING, GONG ZHONG-JUN, XU DONG-LI, CHEN XIAO-MING, LIU WEI-WEI, LIN XIN-DA, LI YAN-FEI: "Transcriptome Analysis of the Chinese White Wax Scale Ericerus pela with Focus on Genes Involved in Wax Biosynthesis", PLOS ONE, vol. 7, no. 4, 20 April 2012 (2012-04-20), pages 1 - 10, XP055870168, DOI: 10.1371/journal.pone.0035719
LIU BO-WEN;WANG XUE-QING;SUN TAO;QI QIAN;YU SHU-HUI;YANG PU;CHEN XIAO-MING: "Cloning and Prokaryotic Expression of Wax Synthase Gene of the Chinese White Wax Scale", FOREST RESEARCH, vol. 29, no. 4, 15 August 2016 (2016-08-15), CN, pages 610 - 614, XP055870173, ISSN: 1001-1498, DOI: 10.13275/j.cnki.lykxyj.2016.04.021
YANG PU, ZHU JIA-YING, GONG ZHONG-JUN, XU DONG-LI, CHEN XIAO-MING, LIU WEI-WEI, LIN XIN-DA, LI YAN-FEI: "Transcriptome Analysis of the Chinese White Wax Scale Ericerus pela with Focus on Genes Involved in Wax Biosynthesis", PLOS ONE, vol. 7, no. 4, 20 April 2012 (2012-04-20), pages 1 - 10, XP055870168, DOI: 10.1371/journal.pone.0035719
LIU BO-WEN;WANG XUE-QING;SUN TAO;QI QIAN;YU SHU-HUI;YANG PU;CHEN XIAO-MING: "Cloning and Prokaryotic Expression of Wax Synthase Gene of the Chinese White Wax Scale", FOREST RESEARCH, vol. 29, no. 4, 15 August 2016 (2016-08-15), CN, pages 610 - 614, XP055870173, ISSN: 1001-1498, DOI: 10.13275/j.cnki.lykxyj.2016.04.021
Attorney, Agent or Firm:
KUNMING ZHENGYUAN PATENT & TRADEMARK AGENCY CO., LTD (CN)
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