白背飞虱细胞色素P450还原酶基因的分子特征与表达模式分析

余航， 刘苏， 朱晴子， 周文武， 梁庆梅， 史肖肖， 祝梓杰， 祝增荣

(浙江大学昆虫科学研究所/水稻生物学国家重点实验室/农业部农业昆虫学重点实验室，杭州 310058)



白背飞虱细胞色素P450还原酶基因的分子特征与表达模式分析

余航， 刘苏， 朱晴子， 周文武， 梁庆梅， 史肖肖， 祝梓杰， 祝增荣*

(浙江大学昆虫科学研究所/水稻生物学国家重点实验室/农业部农业昆虫学重点实验室，杭州 310058)

从水稻重要害虫白背飞虱(Sogatellafurcifera)中克隆了一条编码细胞色素P450还原酶(cytochrome P450 reductase，CPR)的全长cDNA，命名为SfCPR，其开放阅读框全长为2 034 bp，编码一个含有677个氨基酸残基的蛋白质。SfCPR蛋白质的2级结构具有CPR的典型特征，例如N端跨膜区、黄素单核苷酸结合域、黄素腺嘌呤二核苷酸结合域和烟酰胺腺嘌呤二核苷酸磷酸结合域，以及保守的催化残基。系统进化分析结果显示，SfCPR与灰飞虱和褐飞虱的CPRs聚在同一分支。荧光定量聚合酶链式反应结果显示：SfCPR基因在白背飞虱各龄期均有表达，其中以成虫表达量最高；在白背飞虱成虫各组织中均能够检测到SfCPR基因的表达，其中，以腹部表达量最高。使用亚致死剂量的溴氰菊酯、吡虫啉和噻嗪酮处理白背飞虱3龄若虫后，试虫的SfCPR表达水平均显著上升。上述结果为进一步研究该基因的生理功能奠定了基础。

细胞色素P450还原酶； 白背飞虱； 杀虫剂； 表达模式

Summary Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is an electron supplier for various cytochrome P450 monooxygenases. Most P450-mediated catalytic reactions in insects require involvement of CPR, such as detoxification of insecticides and plant secondary metabolites. So far, CPR genes have been identified and characterized from many model and non-model insect species. Since insect P450 system is one of the most important metabolic adaptive traits involved in the degradation of xenobiotics and regulation of endogenous substrates. CPR, as the indispensable electron donor of P450 system, has attracted increasing attention as a potential candidate to develop novel chemical inhibitor to manage target insect pests. Rice planthoppers, such asNilaparvatalugens,LaodelphaxstriatellusandSogatellafurcifera, are considered to be the most serious pests of rice. Previously, some studies onL.striatellusandN.lugensfound that silencing the CPR gene by RNAi technology could increase their sensitivity to insecticides, but little was known aboutS.furcifera. This work firstly reports the identification of CPR gene inS.furciferaand up-regulation of the CPR transcription by chemical insecticides. The research will facilitate further study on the function of CPR inS.furcifera.

In this study, a full-length cDNA encoding CPR was cloned fromS.furcifera. The phylogenetic relationships ofSfCPRwith other insect CPRs were estimated by neighbor-joining method, and its distribution in various tissues and different developmental stages were analyzed by real-time quantitative polymerase chain reaction (qPCR). Finally, after exposure of deltamethrin, buprofezin and imidacloprid at sublethal concentrations for 6, 12 and 24 h, the relative expression levels ofSfCPRin the third-instar nymphs were investigated.

By searching the transcriptome data sets ofS.furcifera, a cDNA fragment encoding putative CPR (namedSfCPR) was identified. This cDNA fragment was then amplified by PCR and sequenced in order to confirm that the sequence was not chimeric. TheSfCPRcDNA contained a complete open reading frame (ORF) of 2 034 bp nucleotides, encoding a protein of 677 amino acid residues. The theoretical isoelectric point (pI) and calculated molecular mass of SfCPR protein are 5.48 and 76.762 ku, respectively. The secondary structure of SfCPR protein showed the marked features of typical CPRs, such as N-terminal transmembrane region, FMN-, FAD- and NADPH-binding domains and conserved catalytic residues. In addition, a transmembrane anchor was predicted in the N-terminus of the protein, indicating that SfCPR is an endoplasmic reticulum-located protein. Phylogenic analysis was used to gain insight into the phylogenetic relationships among CPR proteins from diverse insect species. We found that SfCPR and CPRs from other two planthoppers were clustered together. Real-time quantitative PCR showed that the expression ofSfCPRwas detectable in all developmental stages and the level in adults was the highest. TheSfCPRtranscripts were also expressed in all the tested tissues of the adults, and most were strongly expressed in the abdomen. The exposure at sublethal concentrations of deltamethrin, buprofezin and imidacloprid led to significantly elevated expression ofSfCPR. The expression ofSfCPRwas activated soon (6 h) after treatment with buprofezin and imidacloprid, while the response ofSfCPRexpression to deltamethrin was relatively slow (12 h).

In conclusion, this work is the first report about the cDNA sequence information, secondary structure and transcription profiles of CPR gene in theS.furcifera. These findings provide foundation for further research on the physiological function ofSfCPR.

烟酰胺腺嘌呤二核苷酸磷酸-细胞色素P450还原酶(cytochrome P450 reductase，CPR)是细胞色素P450单加氧酶(cytochrome P450 monooxygenases，CYP)的电子供体，在P450介导的诸多催化反应中起着非常重要的作用[1]。对于昆虫而言，大多数P450介导的对杀虫剂和植物次生代谢物的解毒过程中都需要有CPR的参与[2-3]。……