关键词: 沙葱萤叶甲, 表皮蛋白, 基因克隆, 生物信息学, 表达谱, RT-qPCR

Abstract: 【Aim】 Cuticular proteins, the main components of insect integument, play an important role in the development of insects. This study aims to identify cuticular protein genes from Galeruca daurica and to analyze their expression patterns, so as to lay a necessary foundation for investigation on their functions in the growth and development of G. daurica. 【Methods】 The complete open reading frame (ORF) sequences of cuticular protein genes were identified from G. daurica by bioinformatics methods according to the sequence information available in the transcriptome data assembled by our laboratory, and the expression profiles of the eight cuticular protein genes identified at different developmental stages and in different tissues (head, integument, digestive tract and fat body) of the 3rd instar larvae were detected by RT-qPCR. 【Results】 The complete ORF sequences of eight cuticular protein genes were identified from the transcriptome data of G. daurica, and named GdauCP1-8 (GenBank accession numbers: MN629000-MN629007). The ORFs of GdauCP1-8 are 417-810 bp in length, encoding eight proteins of 138-269 amino acids with the predicted molecular weight of 15-28 kD and pI of 4.45-8.62. The encoded proteins have signal peptides with 16-20 amino acids. GdauCP1 has a typical transmembrane domain, while the other seven GdauCPs do not have. Homologous sequence alignment and phylogenetic analysis showed that GdauCP3 has the highest amino acid sequence identity (60.00%) with Leptinotarsa decemlineata CP, while the other GdauCPs have the highest amino acid sequence identities (58.52%-80.00%) with Diabrotica virgifera virgifera CPs. GdauCP1-4 belong to the subfamily RR-2, GdauCP5-7 belong to the subfamily RR-1, and the subfamily identity of GdauCP8 was not defined. The RT-qPCR results showed that all of the eight GdauCP genes were significantly differentially expressed at different development stages and in different tissues of the 3rd instar larvae of G. daurica. GdauCP2, GdauCP4, GdauCP5 and GdauCP6 were highly expressed in the 1st instar larva, GdauCP3, GdauCP7 and GdauCP8 had high expression levels in the pupa, and GdauCP1 was highly expressed in the day-3 3rd instar larva. Except for the higher expression level of GdauCP2 in the adult, the other GdauCP genes had lower expression levels in the adult. GdauCP1 was highly expressed in the head and integument, and GdauCP2 and GdauCP8 had high expression levels in the fat body. GdauCP3, GdauCP4, GdauCP6 and GdauCP7 were highly expressed in the digestive tract, while GdauCP5 was highly expressed in the integument. 【Conclusion】 The expression levels of the eight GdauCP genes significantly differ in different developmental stages and larval tissues of G. daurica with different expression patterns, implying that different GdauCPs may have distinct functions.

Key words: Galeruca daurica, cuticular protein, gene cloning, bioinformatics, expression profiling, RT-qPCR