马铃薯甲虫两个系统性RNA干扰缺失基cDNA的克隆、序列分析和时空表达
Molecular cloning, sequence analysis and expression profiles ofLdeSid-1a and LdeSid-1c genes in Leptinotarsa decemlineata (Say)
杨 帅1** 付开赟2 吴家和3 李国清4 吐尔逊2 郭文超2***
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DOI：10.7679/j.issn.2095-1353.2016.046
作者单位：1. 石河子大学农学院，石河子 832003；2. 新疆农业科学院植物保护研究所，乌鲁木齐 830091；3. 中国科学院微生物研究所植物基因组学国家重点实验室，北京 100101;4. 南京农业大学植物保护学院农作物生物灾害综合治理教育部重点实验室，南京 210095
中文关键词：马铃薯甲虫，系统性RNA干扰缺失基因，时空表达，定量PCR，mRNA水平
英文关键词：Leptinotarsa decemlineata, Sid-1, spatial and temporal expression pattern, real-time quantitive PCR, mRNA transcrpt level
中文摘要：
【目的】 克隆马铃薯甲虫Leptinotarsa decemlineata (Say) 两条Sid-1基因的全长并分析其时空表达。【方法】 本研究通过RT-PCR和5¢/3¢ RACE等技术从马铃薯甲虫中克隆全长序列，通过序列多重比对和系统发育分析研究序列的保守性和基因起源，通过阶段收样，组织解剖和qPCR技术获得这两个基因的时空表达。【结果】 从马铃薯甲虫Leptinotarsa decemlineata的4龄幼虫中克隆得到LdeSid-1a和LdeSid-1c，其mRNA全长为2 887和3 733 bp，编码759和782个氨基酸，分子量为86.19和90.27 ku，两条蛋白质序列的相似性为59%。与其它昆虫的系统性RNA干扰缺失基因的比对结果显示，LdeSid-1a和LdeSid-1c分属于鞘翅目系统性RNA干扰缺失基因的两个分支，均具有典型的11个跨膜域结构，在蛋白质序列的N端具有4段高度保守的基序。qPCR的时序分析表明LdeSid-1a和LdeSid-1c的表达从初孵幼虫开始逐渐上升，而LdeSid-1c在卵中的表达也较高，组织中的分布结果表明，两个基因在所有组织中均表达，在前肠、中肠和后肠以及生殖系统中表达较高，在神经系统中为优势表达。LdeSid-1a和LdeSid-1c的GenBank登录号为KR153284和KR153285。【结论】 LdeSid-1a和LdeSid-1c具有典型的系统性RNA干扰缺失基因家族的结构，时空表达和系统发育的结果均表明这两条基因可能在幼虫的高龄阶段起到重要作用。
英文摘要：
[Objectives] To clone and analyze two Sid-1 full coding sequences and expression profiles in Leptinotarsa decemlineata. [Methods] Full coding sequences of two Sid-1s were amplified by RT-PCR and RACE-PCR, conserved motifs and origin of Sid-1s were analyzed by multiple alignment and construction of phylogenetic trees, and temporal and spatial expression levels detected by qPCR. [Results] A full Open Reading Frame (ORF) of LdeSid-1a and LdeSid-1c were cloned by RT-PCR and 5¢/3¢ RACE from 4th instar larvae of the Colorado Potato Beetle, Leptinotarsa decemlineata. The respective mRNA fragments were 2 887 and 3 733 base pairs in length, encoding proteins comprised of 759 and 782 amino acids with a predicted molecular mass of approximately 86.19 and 90.27 ku. Similarity of these two proteins was 59%. Alignment of LdeSid-1a and LdeSid-1a with Sid-1s of other insects indicates that LdeSid-1a and LdeSid-1c are members of Coleptera Sid-1s family. LdeSid-1a and LdeSid-1c possess 11 transmembrane structures and 4 highly conserved motifs at the N-terminal. The temporal expression pattern of LdeSid-1a and LdeSid-1c determined by qPCR shows that expression of these two genes gradually increases as larvae grow; LdeSid-1c is highly expressed in the egg stage. Spatial expression patterns indicate that these two genes were expressed in all tested tissues; moderately expressed in the digestive system, including the foregut, midgut and hindgut, and predominantly expressed in the nervous system. The LdeSid-1a and LdeSid-1c sequences have been deposited with GenBank under Accession Numbers KR153284 and KR153285. [Conclusion] LdeSid-1a and LdeSid-1c have typical Sid-1 structure and expression patterns, and phylogenetic analysis indicate these two genes may play key roles in larval development.