家蚕sirtuin家族基因的鉴定及系统发生与表达芯片分析

doi:10.3864/j.issn.0578-1752.2014.13.018

摘要/Abstract

摘要： 【目的】鉴于sirtuin家族基因重要而多样性的功能，从家蚕全基因组中鉴定sirtuin家族基因，并进行基因结构、蛋白结构及其理化性质、基因进化、组织表达芯片分析及在不同组织中的定量表达情况分析，为研究家蚕sirtuin家族基因的功能和推动家蚕模式生物系统化的研究奠定基础。【方法】基于家蚕基因组数据库，通过生物信息学手段，利用比较基因组学方法，鉴定家蚕sirtuin家族成员；开放阅读框（ORF）的预测用ORFfinder进行；使用SIM4进行ORF的内含子和外显子的预测；用GSDS和ExPaSy在线工具分别进行基因结构图和蛋白序列理化性质的预测；用CLUSTAL_X软件进行多序列联配及对其二级结构进行分析，并用ESpript进行二级结构作图；使用SMART在线软件进行蛋白功能域预测；MEGA5.0软件用于系统发生树分析；利用已有的家蚕幼虫5龄第3天的芯片数据进行组织表达分析；利用荧光定量PCR技术检测家蚕sirtuin家族基因在5龄第3天幼虫不同组织中的表达情况。【结果】系统分析鉴定了家蚕中存在的5个sirtuin家族基因（Bmsirt2、Bmsirt4、Bmsirt5、Bmsirt6、Bmsirt7），共分为4类（I、II、III、IV）。5个基因分布在家蚕5条染色体上，均为单拷贝基因。基因结构分析显示5个基因均为多外显子基因。同源比对和系统进化分析发现家蚕的sirtuin家族基因与类群中其他昆虫的同源基因形成明显的直系同源关系且高度同源，同样不含有sirt3。蛋白结构预测发现家蚕的sirt6与其他物种的sirt6蛋白一样含有两个sir2结构域聚在一起。组织表达芯片分析发现，sirtuin家族基因在多个组织中具有转录活性。荧光定量PCR检测结果显示家蚕的Bmsirt4在脂肪体、丝腺中低表达；Bmsirt5在精巢、中肠中高量表达，在脂肪体、血液、丝腺中低量表达，与芯片数据基本一致。【结论】通过全基因组分析，家蚕共有5个sirtuin家族成员，进化上分为4类，且与其他昆虫高度同源，芯片数据和实时荧光定量 PCR 结果基本一致，分析表明组织表达模式具有多样性。

关键词: sirtuin , 生物信息学 , 表达特征 , 家蚕

Abstract: 【Objective】The objectives of this study are to identify sirtuin genes from silkworm genome, analyze the gene structure, protein structure, physicochemical properties, phylogeny and tissue expression pattern, and further to provide a basis for the function of sirtuins of silkworm and silkworm model organismal system.【Method】Based on silkworm genome database and bioinformatic method, silkworm sirtuin family genes were identified by comparative genomic analysis. The opening reading frame (ORF) of sirtuins was predicted by ORFfinder, exon and intron were predicted by SIM4. Gene structure was created using GSDS. Physicochemical properties of sirtuin proteins were done by ExPaSy server, multiple sequence alignment and its secondary structure analysis were done by CLUSTAL_X and ESpript, protein structure and phylogenetic tree were analyzed using SMART and MEGA5.0 software. The expression pattern of sirtuin genes in different tissues was analyzed based on the existing microarray database of 3-day-old 5th instar larvae. The expression level of sirtuin genes of silkworm in different tissues of 3-day-old 5th instar larvae were determined by quantitative RT-PCR.【Result】A total of 5 sirtuin genes (Bmsirt2, Bmsirt4, Bmsirt5, Bmsirt6, Bmsirt7) were systematically identified from silkworm and classified into 4 classes (I, II, III, IV). These genes were distributed on 5 chromosomes, and they were single copy genes. Homologous alignment and phylogenetic analysis revealed that the sirtuin genes in silkworm had apparent orthologous relationship with homologous genes from other insects respectively and they were highly homologous, also lack of sirt3. Protein structures showed that sirt6 had two sir2 domain associated together, as the same as other organisms. Microarray analysis showed that the sirtuin family genes had transcriptional active in multiple tissues. Analysis by real time quantitative PCR indicated that Bmsirt4 was expressed relatively lower abundance in fatboby and silk gland. Bmsirt5 was expressed relatively higher abundance in testis and midgut, but was lower abundance in fatboby, hemocyte, and silk gland. This was consistent with the microarray data. 【Conclusion】 Five genes of sirtuin family in silkworm were identified by genome-wide screening, they were classified into 4 classes, highly homologous with other insects. Microarray data was consistent with the result of real-time fluorescence quantitative PCR, and the analysis showed that the tissue expression pattern was diversity.

Key words: sirtuin , bioinformatics , expression characteristics , Bombyx mori

陈聪, 宋江波, 孟刚, 童晓玲, 代方银, 鲁成. 家蚕sirtuin家族基因的鉴定及系统发生与表达芯片分析[J]. 中国农业科学, 2014, 47(13): 2659-2670.

CHEN Cong, SONG Jiang-Bo, MENG Gang, TONG Xiao-Ling, DAI Fang-Yin, LU Cheng. Identification, Phylogenetic and Tissue Expression Microarray Analyses of the Sirtuin Gene Family in Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2014, 47(13): 2659-2670.

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