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根据秀丽小杆线虫Caenorhabditis elegans及其他昆虫中糖基转移酶基因的同源序列,采用逆转录PCR(reverse transcription PCR,RT-PCR)和cDNA末端快速扩增PCR(rapid amplification of cDNA ends PCR,RACE-PCR)技术从小菜蛾Plutella xylostella4龄幼虫中肠组织中获得了3个基因的全长序列,其开放阅读框长度分别为1383bp,1230bp和1041bp,分别编码460,409和346个氨基酸,将其分别命名为Pxbre-3,Pxbre-4和Pxbre-5(GenBank登录号分别是GU321343,GU321344和GU321345)。同源序列比对发现,这些基因和线虫中相应的糖基转移酶基因具有较高相似度,且具有对糖基转移酶活性起关键作用的氨基酸保守模块,也包含类似的跨膜区。由此推测,这3个基因为小菜蛾糖基转移酶基因,参与小菜蛾鞘糖脂的合成。实时定量PCR检测结果表明,这3个基因以及Pxbre-2在小菜蛾4龄幼虫、蛹和成虫以及4龄幼虫中肠组织均有表达,在蛹和成虫阶段表达量较高。本实验结果为今后研究鞘糖脂是否参与小菜蛾对Bt毒素的抗性奠定了基础。
According to the homologous sequences of the glycosyltransferase genes in Caenorhabditis elegans and other insects, the reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) -PCR), the full-length sequence of three genes was obtained from the midgut tissues of the 4th instar larvae of Plutella xylostella. The open reading frames (ORFs) were 1383bp, 1230bp and 1041bp, encoding 460, 409 and 346 amino acids, respectively, Pxbre-3, Pxbre-4 and Pxbre-5 (GenBank accession numbers GU321343, GU321344 and GU321345, respectively). Homologous sequence alignment revealed that these genes are highly similar to the corresponding glycosyltransferase genes in nematodes and have amino acid conserved blocks that play key roles in glycosyltransferase activity and also contain similar transmembrane domains. Therefore, it is speculated that these three genes are the diamondback moth glycosyltransferase gene involved in the synthesis of Plutella xylostella lipids. The results of real-time quantitative PCR showed that the three genes and Pxbre-2 were expressed in midgut tissues of 4th instar larvae, pupal and adult as well as 4th instar larvae of Plutella xylostella, and higher in pupa and adult stages. The results of this study for the future study of glycosphingolipid whether or not to participate in the diamondback moth Bt toxin resistance laid the foundation.