昆虫学报 ›› 2020, Vol. 63 ›› Issue (9): 1059-1069.doi: 10.16380/j.kcxb.2020.09.003

• 研究论文 • 上一篇    下一篇

沙葱萤叶甲钙结合蛋白基因的鉴定及表达谱分析

李爽1, 李玲1, 周晓榕1, 庞保平1,*, 单艳敏2   

  1. (1. 内蒙古农业大学草原昆虫研究中心, 呼和浩特 010020; 2. 内蒙古自治区草原工作站, 呼和浩特 010020)
  • 出版日期:2020-09-20 发布日期:2020-09-30

Identification and expression profiling of calcium-binding protein genes in Galeruca daurica (Coleoptera: Chrysomelidae)

LI Shuang1, LI Ling1, ZHOU Xiao-Rong1, PANG Bao-Ping1,*, SHAN Yan-Min2   

  1.  (1. Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010020, China; 2. Grassland Station of Inner Mongolia Autonomous Region, Hohhot 010020, China)
  • Online:2020-09-20 Published:2020-09-30

摘要: 【目的】沙葱萤叶甲Galeruca daurica是一种近年来在内蒙古草原上猖獗成灾的新害虫。本研究旨在克隆沙葱萤叶甲钙结合蛋白(calcium-binding protein, CaBP)基因,分析其在沙葱萤叶甲成虫不同发育阶段及不同温度下的表达谱,为进一步探究其在沙葱萤叶甲生长发育及滞育过程中的作用奠定基础。【方法】根据沙葱萤叶甲转录组和蛋白质组数据,筛选CaBP基因序列信息,应用RT-PCR技术克隆获得CaBP基因的开放阅读框(ORF)全长序列,并对其进行生物信息学分析;通过qPCR检测其在沙葱萤叶甲成虫不同日龄(羽化后3, 7, 10, 15, 20, 30, 40, 60, 90和110 d)及3日龄成虫在不同温度(0, 5, 10, 15, 20, 25, 30和35℃)下处理1 h后的表达水平。【结果】克隆得到4条具有完整ORF的沙葱萤叶甲CaBP基因cDNA序列,分别命名为GdCaM, GdCAPSL, GdTnClGdCRT(GenBank登录号: MN695412-695415),ORF全长分别为480, 648, 516和1 209 bp,分别编码149, 215, 171和402个氨基酸;只有GdCRT拥有信号肽。同源序列比对和系统发育分析表明,GdCaM, GdCAPSL, GdTnCl和GdCRT分别与玉米根萤叶甲Diabrotica virgifera virgifera的CaM, CAPSL, TnCl及马铃薯甲虫Leptinotarsa decemlineata CRT的氨基酸序列一致性最高,分别为100.0%, 74.0%, 88.2%和92.5%。qPCR结果表明,4个CaBP基因在沙葱萤叶甲不同日龄成虫中均差异表达,且表达模式不同。GdCaM在成虫滞育前(羽化后3 d)表达量较高,进入滞育后(羽化后7 d)表达量降低,在滞育期间(羽化后7-60 d)表达量变化较小,而解除滞育后(羽化后90 d)表达量进一步下调。GdCAPSL在滞育初期(羽化后10 d)维持在最低水平,进入滞育中后期(羽化后40和60 d)开始回升,滞育解除后又突然下调至最低水平,而羽化后110 d急剧上升至最高水平。GdTnCl在滞育初期(羽化后15-20 d)高表达,进入滞育中后期(羽化后30-60 d)急剧下降至最低水平,滞育解除后再次上调,但羽化后110 d突然下调至最低水平。GdCRT在进入滞育后表达量开始逐渐下调,在滞育维持期间(羽化后15-60 d)维持在低水平,滞育解除后又开始上升。温度对3日龄成虫中除GdCRT外的其他3个CaBP基因的表达有显著影响。温度低于20℃时,GdCaM的表达量随温度的降低而升高,但0℃时突然下降;温度高于20℃时,GdCaM的表达量随着温度的升高而上升。GdCAPSL的表达量随着温度的升高而呈现上升的趋势,25℃时达到最高,然后下降。GdTnCl随着温度的升高,表达量呈现下降的趋势。【结论】钙结合蛋白可能在沙葱萤叶甲成虫生长发育及夏滞育调控过程中发挥着重要作用。

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, GdCAPSLGdTnCl 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