小菜蛾鞣化激素基因克隆及其功能分析

Cloning and functional analysis of bursicon genes in the diamondback moth,Plutella xylostella (Lepidoptera:Plutellidae)

鞣化激素是调节昆虫表皮骨化和翅膀发育的一种神经激素，尽管已经在许多不同种昆虫上克隆了鞣化激素基因，但是关于小菜蛾Plutella xylostella鞣化激素及其基因的研究至今未见报道。本研究克隆了两个小菜蛾鞣化激素基因Pxbursα和Pxbursβ（GenBank登录号分别为KF498645和KF498646）全长cDNA，其序列长度分别为537bp和360bp，与已报道的其他昆虫的鞣化激素氨基酸序列一致性分别为51％～68％和37％～57％。实时定量PCR分析发现Pxbursα和Pxbursβ均在蛹期表达量高，而在幼虫期和成虫期的表达量低。以Pxbursα部分序列的双链RNA（dsRNA）饲喂小菜蛾4龄末期幼虫，发现蛹期Pxbursα的表达受到了显著抑制，小菜蛾的发育停滞在蛹期而无法正常羽化，并最终死亡。由此推测，小菜蛾鞣化激素基因在蛹期的大量表达对其生长发育和羽化具有重要的作用。

Bursicon is a neurohormone that regulates cuticle sclerotization （tanning and hardening） and the wing expansion processes in insects. Although bursicon genes have been well characterized from several insect species or predicted from insect genomes, bursicon and its genes in the diamondback moth （DBM） , Plutella xylostella, remain poorly understood. In this study, two bursicon genes, Pxbursα and Pxbursβ （GenBank accession numbers： KF498645 and KF498646, respectively）, were cloned from P. xylostella with the full-length cDNA of 537 bp and 360 bp, respectively. The nucleotide sequence identities of Pxbursot and Pxbursfl with the bursicon genes from other insect species range from 51% to 68%, and 37% to 57% , respectively. Based on qRT-PCR results, we found that bursicon genes were expressed at a higher level in pupae, but at lower levels in larvae and adults. When the 4th-instar larvae were fed with double-stranded RNA of Pxbursα, the expression of Pxbursα in pupae subsequently declined significantly. Thus, the development of pupae was seriously delayed, and pupae died before eclosion. We inferred that higher level expression of Pxburs genes in pupae might play a key role in the metamorphosis and wing expansion of P. xylostella.