椰心叶甲CYP4基因的克隆及绿僵菌侵染后的诱导表达研究

根据细胞色素P450家族4（CYP4）的氨基酸保守序列设计1对简并引物，从椰心叶甲Brontispa longissima成虫总RNA中扩增得到5个cDNA片段（GenBank登录号: DQ238840－DQ238844）。以3′-RACE法获得片段BLWH4的3′端序列，推导的氨基酸序列表明其结构中含有CYP家族的特征性保守序列： 螺旋K区的ETLR和血红素结合区的F××G×××C×G。以18S 为对照的RT-PCR分析表明，BLWH4在成虫的mRNA表达量远大于幼虫。绿僵菌Metarhizium anisopliae菌株MA-3和MA-4侵染椰心叶甲成虫及5龄幼虫后，BLWH4的mRNA表达增强，提示BLWH4可能具有增强椰心叶甲抵抗绿僵菌侵染的作用。

Identification and induction by Metarhizium anisopliae infection of family 4 cytochrome P450 in Brontispa longissima

In order to obtain cDNA clones coding for CYP4 proteins in Brontispa longissima, degenerate primers were designed using the conserved sequences of known CYP4s and used to amplify partial cDNA fragments from adults B. longissima. Five genes were identified (GenBank accession numbers: DQ238840 －DQ238844), and the 3′-end of BLWH4 was cloned by 3′-RACE. The deduced amino acid sequence of BLWH4 has CYP conserved domains of helix K and heme-binding motif. The RT-PCR analysis indicated that the BLWH4 mRNA expression in adults was more than in larvae, and increased when adults and 5th instar larvae were infected with Metarhizium anisopliae MA-3 and MA-4. The results suggested that BLWH4 should play a role in protection against pathogenic fungus.