绵羊TGFβ2基因及其剪切体的克隆与生物信息学分析

Cloning and Bioinformatic Analysis of TGFβ2 and Its Spliced Variant

旨在克隆绵羊TGFβ2(Transforming growth factor-β2)基因CDS,解析其序列及编码蛋白的生物学特性,为绵羊生产利用提供理论基础。采用PCR技术克隆获得TGFβ2基因的CDS,并进行生物信息学分析。结果表明:绵羊TGFβ2基因的CDS长度为1 329bp,编码442个氨基酸;其可变剪切体TGFβ2-AS1和TGFβ2-AS2由于CDS部分缺失分别编码331和414个氨基酸。绵羊TGFβ2及其可变剪切体TGFβ2-AS1和TGFβ2-AS2编码蛋白的理论等电点分别为8.74、7.60和8.82,均存在1个信号肽和1个跨膜结构域,由α-螺旋、β-折叠、延伸及无规则卷曲构成;序列同源性分析发现,绵羊TGFβ2基因编码区的核苷酸和氨基酸序列与其他哺乳动物同源性较高,进化树分析表明,绵羊TGFβ2氨基酸序列与人类的进化关系较近,与斑马鱼较远;细胞定位分析表明TGFβ2及其可变剪切体TGFβ2-AS1和TGFβ2-AS2主要在细胞外基质中发挥作用,调控下游靶基因的表达。获得绵羊TGFβ2及其可变剪切体TGFβ2-AS1和TGFβ2-AS2完整的CDS,与TGFβ2转录本相比,TGFβ2-AS1和TGFβ2-AS2分别缺失111和28个氨基酸序列,其可能通过与TGFβ2转录本不同的分子调控机制发挥重要的生物学作用。

The objective of this study is to reveal the biological property of TGFβ2 gene in sheep and to provide the theoretical data for application by cloning TGFβ2 coding region sequence （CDS） and analyze its sequence as well as genetic features. The CDS of sheep TGFβ2 was cloned using RT-PCR technique,and then,its genetic characteristics were analyzed by bioinformatics methods. The results showed that the encoding sequence of sheep TGFβ2 gene was 1 329 bp,encoding 442 amino acids;due to partial deletion of sequences,the variant spliced TGFβ2-AS1 and TGFβ2-AS2 encoding respectively encoded 331 and 414 amino acids. Sheep TGFβ2 and variable isoform TGFβ2-AS1 and TGFβ2-AS2 protein isoelectric point were 8.74,7.60 and 8.82 respectively,which had a signal peptide and a trans- membrane domain and contained alpha helix, beta- fold, extension and random curl;the analysis of se- quence homology found that the nucleotide and amino acid sequence of sheep TGFβ2 CDS had higher homology with other mammals,the phylogenetic tree analysis indicated the sheep TGFβ2 was closely related to human, but distantly related to the zebrafish; cell location analysis showed that TGFβ2 and its variable shear body structure played a biological role in extracellular matrix, and regulate down- stream target gene expression. This research obtained the TGFβ2 and its spliced variant TGFβ2-ASI and TGFβZ-AS2 of complete encod region sequence. However,compared with TGFβ2 transcript, 111 and 28 amino acid sequences were missing from TGFβ2-AS1 and TGFβ2-AS2, respectively, which it probably involved in different physiological functions.