褐色橘蚜RR-2型表皮蛋白基因鉴定及功能分析

Identification and function analysis of RR-2 CPR genes in brown citrus aphid Toxoptera citricida(Kirkaldy)

为明确褐色橘蚜Toxoptera citricida RR-2型表皮蛋白基因的数量、表达特性及其在发育中的潜在功能,运用转录组数据和生物信息学软件对其进行鉴定和分析,利用实时荧光定量PCR技术测定其在褐色橘蚜不同发育阶段、有翅/无翅成蚜及不同组织中的表达量,并利用RNAi方法探索其在褐色橘蚜生长发育过程中的潜在功能。结果显示,共筛选鉴定出45个CPR(cuticular protein with R&R Consensus)家族基因,其中33个有完整的开放阅读框,具RR-2型CPR家族特有的签名序列及与几丁质结合必需的酪氨酸和苯丙氨酸残基,推测这些序列编码的RR-2型CPR具有几丁质结合活性。系统发育分析表明大部分褐色橘蚜RR-2型CPR同源性较高。TcCPR10、TcCPR14、TcCPR28和TcCPR68基因在褐色橘蚜若虫蜕皮后表达量较高;TcCPR7、TcCPR10、TcCPR28和TcCPR57基因主要在褐色橘蚜胸腹部表达,在相同组织中TcCPR10和TcCPR68基因在有翅蚜中的表达量显著低于在无翅蚜中的表达量。TcCPR7和TcCPR68基因可被有效沉默,其表达量分别下调26.62%和66.34%,但仅TcCPR68基因的沉默可导致褐色橘蚜大量死亡,存活率仅为36.45%,极显著低于对照组。表明褐色橘蚜RR-2型CPR家族基因的表达具有较强的时空特异性,可能在不同虫态、不同组织中发挥着不同功能;TcCPR68基因可能在褐色橘蚜发育过程中起重要作用。

To identify and evaluate the potential functions of RR-2 cuticular protein genes in brown citrus aphid Toxoptera citricida, bioinfomatic softwares were applied to analysis the data generated from the transcriptome of T. citricida. Quantitative PCR(qPCR) was used to estimate the m RNA expression levels of selected RR-2 CPR(cuticular proteinwith R&R Consensus) genes in different developmental stages, wing types(alate and apterous adult), and body tissues. In addition, RNA interference(RNAi)was applied to evaluate the functions of selected CPR genes. In total, 45 RR-2 CPR genes were identified, including 33 genes with complete open reading frames. The encoded proteins shared a typical R&R Consensus, tyrosine and phenylalanine residues, which indicating their roles in chitin-binding.Phylogenetic analysis showed that, most of the RR-2 CPR genes formed a single clade. The results of q PCR showed that the expression levels of TcCPR10, TcCPR14, TcCPR28 and TcCPR68 were significantly unregulated after molting. TcCPR7, TcCPR10, TcCPR28 and TcCPR57 were mainly expressed in thorax/abdomen, and the expression levels of TcCPR10 and TcCPR68 were highly expressed in apterous adults. The results of RNAi demonstrated that TcCPR7 and TcCPR68 were down regulated for 26.62%and 66.34%, respectively. The survival rate of T. citricida decreased to 36.45% after interference with TcCPR68. This study indicated that the RR-2 CPR genes might had a distinct roles in different conditions, and TcCPR68 might involve in the development of T. citricida.