关键词: 沙葱萤叶甲, 钙结合蛋白, 基因克隆, 表达谱分析, 夏滞育, 温度

Abstract: 【Aim】 Galeruca daurica is a new pest with great outbreaks in grasslands in Inner Mongolia in recent years. This study aims to clone the calcium-binding protein (CaBP) genes from G. daurica and to analyze their expression profiles at different developmental stages of the adults and under different temperatures, so as to lay a foundation for further study on their roles in the growth, development, and diapause of G. daurica. 【Methods】 The full-length ORFs of CaBP genes were cloned from G. daurica by RT-PCR according to the sequence information available in the transcriptome and proteome data of G. daurica, and their sequences were subjected to bioinformatics analysis. Their expression profiles in different day-old adults (3, 7, 10, 15, 20, 30, 40, 60, 90 and 110 d after eclosion) and in the 3-day-old adults exposed to 0, 5, 10, 15, 20, 25, 30 and 35℃, respectively, for 1 h were detected by qPCR. 【Results】The cDNA sequences with complete ORFs of four CaBP genes were cloned from G. daurica, and named GdCaM, GdCAPSL, GdTnCl and GdCRT (GenBank accession numbers: MN695412-695415), respectively. Their ORFs are 480, 648, 516 and 1 209 bp in length, encoding 149, 215, 171 and 402 amino acids, respectively, and only GdCRT has a signal peptide. Homologous sequence alignment and phylogenetic analysis indicated that GdCaM, GdCAPSL, GdTnC and GdCRT have the highest amino acid sequence identity with CaM, CAPSL and TnCL of Diabrotica virgifera virgifera, and CRT of Leptinotarsa decemlineata, respectively (100.0%, 74.0%, 88.2% and 92.5%, respectively). The qPCR results showed that four CaBPs were differentially expressed in different day-old adults with diverse expression patterns. The expression level of GdCaM was relatively high in adults before diapause (3 d after eclosion), declined in adults beginning to enter diapause (7 d after eclosion), changed faintly during diapause (7-60 d after eclosion), and decreased further after diapause termination (90 d after eclosion). The expression levels of GdCAPSL maintained low in adults during the early diapause stage (10 d after eclosion), began to increase during the middle and late diapause stages (40 and 60 d after eclosion), and suddenly dropped to the lowest after diapause termination but abruptly rose to the highest 110 d after adult eclosion. The expression of GdTnCl was high in adults during the early diapause stage (15-20 d after eclosion), rapidly down-regulated to the lowest levels during the middle and late diapause stages (30-60 d after eclosion), up-regulated again after diapause termination, and suddenly down-regulated to the lowest level 110 d after adult eclosion. The expression levels of GdCRT in adults started to decrease gradually after diapause initiation, kept low during diapause maintenance (15-60 d after eclosion), and began to increase again after diapause termination. Temperature affected significantly the expression levels of the other three CaBP genes except for GdCRT in the 3-day-old adults. The expression levels of GdCaM rose with temperature declining when the temperature was below 20℃, but abruptly dropped at 0℃, while increased with temperature rising when temperature was above 20℃. The expression levels of GdCAPSL had an increasing tendency with temperature increasing, reached the highest at 25℃, and then declined. The expression 
levels of GdTnCl had a decreasing tendency with temperature increasing. 【Conclusion】 CaBPs might play important roles in the growth, development, and regulation of summer diapause of G. daurica adults.

Key words:  Galeruca daurica, calcium-binding protein, gene cloning, expression profiling; summer diapause, temperature