香港牡蛎Foxl2基因克隆分析与表达模式研究

Cloning and Expression Pattern Investigation of Crassostrea hongkongensis Foxl2 Gene

本研究旨在对香港牡蛎Foxl2基因进行克隆、序列分析及建立其在香港牡蛎组织中的表达模式谱。以GenBank数据库中已上传的太平洋牡蛎Foxl2基因序列为模板,设计特异性引物扩增获得香港牡蛎Foxl2基因;应用生物信息学方法,系统分析和预测香港牡蛎Foxl2基因的遗传进化、蛋白质的理化性质和结构特征;并应用QRT-PCR技术对Foxl2在香港牡蛎组织中的表达进行了差异分析。结果表明:香港牡蛎Foxl2基因编码区全长1104 bp,共编码氨基酸367个。多重序列比较分析显示香港牡蛎Foxl2核苷酸序列相似性与太平洋牡蛎、美洲牡蛎、大珠母贝、栉孔扇贝、虾夷扇贝、皱纹盘鲍相应序列的相似性分别为97%、83%、71%、76%、75%、75%,系统进化树分析显示在不同物种以及进化的过程中Foxl2基因具有较高的保守性。Foxl2蛋白质信息学分析结果表明该蛋白呈碱性,N端无信号肽存在,位于胞核中,含有FH等结构。定量分析结果显示:Foxl2在香港牡蛎3种组织中有表达,其中表达量最高的是性腺,其次是唇瓣,鳃中相对表达量最低。本研究成功地克隆了香港牡蛎Foxl2基因,并研究了其在香港牡蛎8种组织中的表达变化规律,为今后阐明其在香港牡蛎性腺发育进程中的功能提供了理论依据。

In this present paper,clone,sequence bioinformatics analysis and expression pattern determination were performed of Foxl2 gene in Crassostrea hongkongensis.A pair of special primers was designed according to the released sequence of Crassostrea gigas Foxl2 in GenBank.The Foxl2 gene was amplified by RT-PCR,its gene sequence character and protein structure was systemically analyzed by bio-informatics techniques.The expression pattern of Crassostrea hongkongensis Foxl2 in different tissues was also assayed with QRT-PCR.The results showed that a 1172 bp Crassostrea hongkongensis Foxl2 gene fragment including a 1104 bp whole length CDS(coded 367 amino acids)was cloned and sequenced.The sequence multi-aligned results showed that Crassostrea hongkongensis Foxl2 gene shared 97%,83%,71%,76%,75%and 75%of similar nucleotide sequence with that of Crassostrea gigas,Crassostrea virginica,Pinctada maxima,Azumapecten farreri,Mizuhopecten yessoensis and Haliotis discus respectively.Foxl2 protein sequence analysis showed that it was weakly alkaline,hadn't signal peptide,positioned in the nuclear,and with the presence of Forkhead structure.In addition,the expression pattern analysis results showed that Crassostrea hongkongensis Foxl2 expressed in gill,gonad and labial palp etc.,with the most abundant expression in gonad,followed by labial palp,the minimal expression in gill was observed.The cloning and analysis of Foxl2 gene provided an important basic theory for further study reproduction regulation mechanism of Foxl2 gene in Crassostrea hongkongensis in the future.