苹果蠹蛾NAPDH-细胞色素P450还原酶（CPR）基因的克隆及表达模式分析 *

doi:10.7679/j.issn.2095-1353.2020.087

摘要/Abstract

摘要：

【目的】 本研究通过克隆苹果蠹蛾Cydia pomonella CPR (CpCPR) 基因cDNA序列,并对该基因的表达模式进行分析,为深入研究P450酶系在苹果蠹蛾对植物次生物质和杀虫剂解毒代谢过程中的作用提供理论基础。【方法】 以近缘昆虫的CPR氨基酸序列作为询问序列,在苹果蠹蛾转录组（SRX371333）中进行筛查比对,获得了苹果蠹蛾CPR（CpCPR）基因的cDNA序列。采用RT-PCR技术克隆目的基因的开放阅读框（ORF）。利用生物信息学软件分析目的基因的序列特征、3D结构和与其他昆虫CPR基因的系统进化关系。采用RT-qPCR技术测定CpCPR基因在苹果蠹蛾不同发育阶段（卵、幼虫、蛹和成虫）及幼虫不同组织部位（头部、表皮、脂肪体、中肠和马氏管）的表达水平。【结果】 克隆获得的苹果蠹蛾CpCPR基因的ORF为2 052 bp,编码683个氨基酸残基,预测的蛋白质分子量（Mw）为77.326 ku,理论等电点（pI）为5.65。CpCPR包含FMN区域、NADPH区域和FAD等昆虫CPR的典型特征。系统发育分析表明,苹果蠹蛾CpCPR与鳞翅目昆虫CPR基因聚在一枝。RT-qPCR结果表明,CpCPR基因在苹果蠹蛾的整个发育阶段均有表达,在幼虫期表达量最高;CpCPR基因在4龄幼虫的各部位均有表达,在中肠中的表达量最高。【结论】 克隆获得了苹果蠹蛾CpCPR基因的ORF序列,该基因在苹果蠹蛾主要取食阶段和消化器官中高表达,表明其可能在苹果蠹蛾对植物次生物质和杀虫剂解毒代谢过程中扮演重要作用。

关键词: 苹果蠹蛾, 细胞色素P450还原酶, 解毒代谢, 抗药性, 基因克隆

Abstract:

[Objectives] In order to improve understanding of the role of cytochrome P450 in the detoxification of plant secondary substances and insecticides, the CPR gene (CpCPR) of the codling moth, Cydia pomonella, was cloned and its expression profile was analyzed. [Methods] The amino acid sequences of CPR genes from closely-related insect species were used as queries to search against the C. pomonella transcriptome database (SRX371333) and obtain the sequence of the C. pomonella CPR gene. RT-PCR was then used to amplify the Open Reading Frame (ORF) of the target gene. Bioinformatics software was used to compare the sequence characteristics, 3D structure and phylogenetic relationship of CpCPR to those of CPR from other insects. Real-time quantitative PCR (RT-qPCR) was used to determine the relative expression level of CpCPR in different developmental stages (eggs, 1-5 instar larvae, pupae, and adults) and in various tissues (head, cuticle, fat body, midgut, and Malpighian tubes). [Results] The length of the CpCPR gene is 20 252 bp, which encodes 683 amino acids with a predicted molecular mass of 77.326 ku and a theoretical isoelectric point of 5.65. CpCPR contains typical insect CPR gene characteristics, such as an FMN region, an NADPH region and a FAD region. Phylogenetic analysis indicates that CpCPR clusters with the CPR gene of other Lepidoptera. RT-qPCR results show that the CpCPR gene is expressed in all developmental stages but is most abundant in the larval stage. In addition, CpCPR mRNA was detected in all tissues but was most abundant in the midgut in fourth-instar larvae. [Conclusion] The ORF of the CpCPR gene was successfully cloned. This gene was most highly expressed during the larval stage and in the midgut of C. pomonella, indicating that it may play an important role in the detoxification of plant secondary substances and insecticides.

Key words: Cydia pomonella, NADPH cytochrome P450 reductase, detoxification, insecticide resistance, gene cloning

李沛蓉, 陈高满, 杨雪清. 苹果蠹蛾NAPDH-细胞色素P450还原酶（CPR）基因的克隆及表达模式分析 ^*[J]. 应用昆虫学报, 2020, 57(4): 850-860.

Pei-Rong LI, Gao-Man CHEN, Xue-Qing YANG. Molecular cloning and expression profiles of NADPH-cytochrome P450 reductase (CPR) gene in the codling moth, Cydia pomonella[J]. Chinese Journal of Applied Entomology, 2020, 57(4): 850-860.

图/表 10

表1 本研究中所用到的引物

Table 1 Primers used in this study

基因Gene	引物序列（5￠-3￠） Primer sequence	用途Purpose
CpCPR-ORF	F：ATGCACGTTTTGAAAATGAGTACAG	ORF克隆 TA克隆测序
CpCPR-ORF	R：CTATGTTCTCGGGACGAAGCGGACA
M13	F：GTTTTCCCAGTCACGAC
M13	R：CAGGAAACAGCTATGAC
CpCPR	F：AGTCTATGAAGTGGGTTTGGG	RT-qPCR
CpCPR	R：GCTCCTCCTCGCCTGTG
β-Actin	F：CGGCATCCACGAAACCACCT
β-Actin	R：TGGAAGGAGCCAGTGCGG

表2 苹果蠹蛾CpCPR蛋白的氨基酸组成

Table 2 The amino acid composition of CpCPR protein in Cydia pomonella

氨基酸 Amino acid	数量 Number	比例（%）Composition	氨基酸 Amino acid	数量 Number	比例（%）Composition
丙氨酸Ala	44	6.44	亮氨酸Leu	67	9.81
精氨酸Arg	31	4.54	赖氨酸Lys	49	7.17
天冬氨酸Asp	43	6.30	甲硫氨酸Met	14	2.05
天冬酰胺Asn	28	4.10	苯丙氨酸Phe	26	3.81
半胱氨酸Cys	11	1.61	脯氨酸Pro	31	4.54
谷氨酰胺Gln	27	3.95	丝氨酸Ser	40	5.86
谷氨酸Glu	55	8.05	苏氨酸Thr	37	5.42
甘氨酸Gly	45	6.59	色氨酸Trp	10	1.46
组氨酸His	18	2.64	络氨酸Tyr	27	3.95
异亮氨酸Ile	37	5.42	缬氨酸Val	43	6.30

图1 苹果蠹蛾CpCPR的疏水性分析

Fig. 1 The hydrophobicity of Cydia pomonella CpCPR

图2 苹果蠹蛾CpCPR蛋白序列跨膜结构预测

Fig. 2 Prediction of the transmembrane structure of Cydia pomonella CpCPR

图3 苹果蠹蛾CpCPR蛋白的信号肽预测

Fig. 3 Prediction of the signal peptide of Cydia pomonella CpCPR

图4 苹果蠹蛾CpCPR与近缘昆虫CPR氨基酸序列对比

Fig. 4 Alignment of the amino acid sequences of CPR from Cydia pomonella and closely-related insect species

图5 苹果蠹蛾CpCPR3D结构的同源建模

Fig. 5 3D structure of CpCPR constructed by using homology modeling

图6 邻接法构建的基于CPR氨基酸序列的苹果蠹蛾和近缘昆虫的系统进化树（1 000次重复）不同的颜色代表不同的昆虫科目,黄色代表鳞翅目,绿色代表双翅目,蓝色代表半翅目;苹果蠹蛾的CPR序列用红色线标注。

Fig. 6 Phylogenetic relationship of Cydia pomonella CpCPR with CPR from other insects amino acid sequence by neighbor-joining method (1 000 replicates) Different colors represent different insects. Yellow, green, and blue represent insects in Lepidoptera, Diptera, and Hemiptera, respectively; The CPR sequence of codling moth is marked with red lines.

图7 苹果蠹蛾不同发育阶段中CpCPR的表达量分析 Egg：卵期;1-5L：分别为1-5龄幼虫;P-M：雄蛹;P-F：雌蛹;A-M：雄成虫;A-F：雌成虫。图中数据为平均值±标准差;柱上标有不同字母表示不同发育阶段基因表达量差异显著（P<0.05, Duncan氏检验法）。下图同。

Fig. 7 Expression profiles of CpCPR in different developmental stages of Cydia pomonnella Egg: Egg; 1-5L: 1st-5th instar larva; P-M: Male pupa; P-F: Female pupa; A-M: Male adult; A-F: Female adult. Data in the figure are mean±SD. Histograms with different letters indicate significant difference in the gene expression among different developmental stages at the 0.05 level by Duncan’s test. The same below.

图8 苹果蠹蛾4龄幼虫不同组织中CpCPR的表达模式分析 HE：头;CU：表皮;FB：脂肪体;MG：中肠;MT：马氏管。

Fig. 8 Expression profiles of CpCPR in different tissues of fourth-instar Cydia pomonnella larvae HE: Head; CU: Cuticle; FB: Fat body; MG: Midgut; MT: Malpighian tubes.

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