Molecular cloning and expression profiles of NADPH-cytochrome P450 reductase (CPR) gene in the codling moth, Cydia pomonella

doi:10.7679/j.issn.2095-1353.2020.087

Abstract

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

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.

Figures/Tables 10

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

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

Fig. 1 The hydrophobicity of Cydia pomonella CpCPR

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

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

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

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

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.

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.

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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