cDNA Library Construction in the Multicolored Asian Ladybird Beetle, Harmonia axyridis (Coleoptera: Coccinellidae) for Gene Functional Analysis Using Gateway Cloning System
In this preliminary study, we sought to investigate the genes involved in the differentiation of elytral color and pattern in the beetle Harmonia axyridis. We confirmed the inhibition of ecdysis after knocking down H. axyridis ecdysone receptor A, and generated an H. axyridis cDNA library using the Gateway system for functional analysis of undiscovered genes in H. axyridis. We generated an H. axyridis. cDNA library with a titer of 4.43 × 104 and insert sizes of approximately 200–700 bp. Examination of the genetic information of this cDNA library showed that most sequences were from insect genes and one was an H. axyridis gene. After cloning H. axyridis ecdysone receptor A into a LITMUS 28i vector, 400–500 bp double– stranded RNA was synthesized using the T7 promoter. When the synthesized double–stranded ecdysone receptor A was injected between the 1st and 2nd sections of the abdomen of 0 to 2–day–old 4th–instar larvae of H. axyridis, most of the larvae died within 4 days after injection, whereas others failed to pupate. qRT– PCR analysis showed that, compared with non–treatment and distilled water treatment groups, the expression level of ecdysone receptor A in larvae injected with double–stranded RNA of ecdysone receptor A decreased markedly at day 4 after injection.
九州大学大学院農学研究院紀要 63(2), 281-292, 2018-09-01
Faculty of Agriculture, Kyushu University