奶牛HSF1基因可变剪接体克隆及生物信息学分析  

Cloning and Bioinformatics Analysis of Alternatively Spliced Variants of HSF1 Gene in Dairy Cattle(Bos taurus)

作　　者：齐颖 张一名[1] 赵梦芸 黄美琦 郭月美 储明星[2] 李秋玲[1] QI Ying;ZHANG Yi-Ming;ZHAO Meng-Yun;HUANG Mei-Qi;GUO Yue-Mei;CHU Ming-Xing;LI Qiu-Ling(Hebei Key Laboratory of Animal Diversity/College of Life Sciences,Langfang Normal University,Langfang 065000,China;Key Laboratory of Animal(Poultry)Genetics Breeding and Reproduction,Ministry of Agriculture and Rural Affairs/Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]廊坊师范学院生命科学学院/河北省动物多样性重点实验室,廊坊065000 [2]中国农业科学院北京畜牧兽医研究所/农业农村部动物遗传育种与繁殖(家禽)重点实验室,北京100193

出　　处：《农业生物技术学报》2021年第9期1722-1732,共11页Journal of Agricultural Biotechnology

基　　金：河北省高等学校科学技术研究项目(QN2020166);农业农村部动物遗传育种与繁殖(家禽)重点实验室开放课题(poultrylab2019-8);河北省高等学校科学技术研究重点项目(ZD2021091)。

摘　　要：热休克转录因子1(heat shock transcription factor 1,HSF1)是启动热休克蛋白基因表达的重要转录因子,对其进行深入研究有助于阐明HSF1对热应激反应的调控机制。本研究采用反转录PCR(reverse transcription-PCR,RT-PCR)技术对中国荷斯坦奶牛(Bos taurus)HSF1基因全长CDS进行克隆,并采用生物信息学技术分析其编码蛋白的生物学特性。结果显示,奶牛HSF1基因的3种剪接体被成功克隆,即HSF1-AS1(GenBank No.MW401766)、HSF1-AS2(GenBank No.XM005215151.4)和HSF1-AS3(GenBank No.MW401767),剪接方式分别为外显子跳跃、内含子保留和可变的3’端位点。开放阅读框分析发现,HSF1的3种剪接体分别编码311、553和517个氨基酸。对本实验室前期的乳腺组织RNA-seq数据进行分析,结果显示,HSF1-AS2剪接体表达量最高(P<0.01),HSF1-AS1表达量最低(P<0.01)。qRT-PCR检测结果显示,热应激使乳腺上皮细胞HSF1-AS1和HSF1-AS2表达水平显著上调。对剪接因子结合位点进行分析,发现HSF1基因第11外显子的SNP(18959 C>T)恰巧处于一个潜在的外显子剪接增强子(exonic splicing enhancer,ESE)区域,可能与HSF1-AS3的形成有关。基因进化分析发现,牛HSF1氨基酸序列与山羊(Capra hircus)同源性较高。本研究为深入阐明HSF1基因表达调控机制提供参考依据。Heat shock transcription factor 1(HSF1)is an important transcription factor that initiates heat shock protein gene expression.Further study on HSF1 will help to clarify the regulatory mechanism of HSF1 on heat stress response.In this study,reverse transcription-PCR(RT-PCR)was used to clone the full-length CDS of HSF1 from Chinese Holstein cow(Bos taurus),and bioinformatics was used to analyze the biological characteristics of the protein.The results showed that 3 splice variants were successfully cloned,named HSF1-AS1(GenBank No.MW401766),HSF1-AS2(GenBank No.XM005215151.4),and HSF1-AS3(GenBank No.MW401767).The splicing patterns of the 3 variants were exon skipping,intron retaining,and alternative 3’splice sites.The ORF analysis showed that the 3 splice variants of HSF1 encoded 311,553 and 517 aa,respectively.The RNA-seq data of breast tissue in previous study was analyzed,and the results showed that the expression of HSF1-AS2 was the highest(P<0.01),and the expression of HSF1-AS1 was the lowest(P<0.01).The results of qRT-PCR showed that the expression levels of HSF1-AS1 and HSF1-AS2 were both up-regulated in heat stressed mammary epithelial cells.The binding site analysis of splicing factors showed that the SNP(18948 C>T)of exon 11 of HSF1 gene was just in the potential region of exonic splicing enhancer(ESE),which might be associated with the formation of HSF1-AS3.The phylogenetic analysis showed that bovine HSF1 amino acid sequence had high homology and close genetic distance with that of goats(Capra hircus).The present study provides a reference for further elucidating the regulation mechanism of HSF1 gene expression.

关 键 词：奶牛 热休克转录因子1基因(HSF1) 可变剪接 热应激 生物信息学 

分 类 号：S823.91[农业科学—畜牧学]