黑线仓鼠Cry1、Cry2克隆及生物信息学分析

Clone and bioinformatics analysis of Cry1、Cry2 in criecetulus barabensis

隐花色素(Cryptochromes,包括Cry1和Cry2)编码黄素腺嘌呤二核苷酸结合蛋白,是昼夜节律核心振荡器复合物的关键组分,是各种生理功能的重要调节因子.本实验以黑线仓鼠(Cricetulus barabensis)为材料,通过设计特异性引物,RT-PCR技术克隆得到黑线仓鼠Cry1 cDNA序列1822 bp(GeneBank登录号:MK796241)、Cry2 cDNA序列1945 bp(GeneBank登录号:MK796242).运用生物信息学对Cry1、Cry2核酸序列以及蛋白质序列进行分析,构建系统进化树,结果表明:测得的Cry1 cDNA包括一个完整的开放阅读框1764 bp,部分5′端非翻译区和部分3′非翻译区,编码587个氨基酸,A+T含量略高于G+C含量;测得的Cry2的cDNA包括一个完整的开放阅读框1794 bp,部分5′端非翻译区和部分3′非翻译区,编码597个氨基酸,G+C含量远高于A+T含量,提示其Tm值较高,热稳定性强.CRY1、CRY2理论蛋白分子量为66469.86 Da、67427.08 Da,等电点(pI)为8.40、8.82,均为不稳定蛋白.CRY1、CRY2蛋白跨膜区和疏水性分析和信号肽分析等均表明,整体疏水性不强,为亲水性蛋白;没有信号肽,不属于分泌蛋白,与其作为转录抑制因子相符合.功能结构域预测表明,二者均含有两个高度保守的功能结构域:DNA光解酶及FAD结合域.同源性比对及系统进化树分析显示,黑线仓鼠与中国仓鼠亲缘关系最近,与灵长类目及鸡形目亲缘关系较远,这与传统的分类物种分类方式相一致,证实本实验测得的Cry1、Cry2基因序列正确,体现了Cry1、Cry2进化的保守性.本实验为探究昼夜节律基因Cry1、Cry2在动物繁殖调控中的作用奠定了基础,具有重要的理论意义.

Cryptochromes(including Cry1 and Cry2)encode a flavin adenine dinucleotide binding protein,which is a key component of the circadian rhythm core oscillator complex and an important regulator of various physiological functions.Based on the striped hamsters(Cricetulus barabensis)for materials.The cDNA sequence of Cry1 and Cry2 are cloned by RT-PCR.The Cry1 sequence is 1822 bp,containing part of the 5′non-coding area,part of the 3′non-coding area and a complete open reading frame of 1764 bp,which encodes a polypeptide of 587 amino acids resides(MK796241).The Cry2 sequence is 1945 bp,containing part of the 5′non-coding area,part of the 3′non-coding area and a complete open reading frame of 1794 bp,which encodes a polypeptide of 597 amino acids resides(MK796242).Contrary to Cry1,the content of G+C is higher than A+T in the Cry2 sequence,indicating that its Tm value is higher and the thermal stability is strong.The theoretical molecular weights of Cry1 and Cry2 are 66469.86 Da and 67427.08 Da,and the isoelectric point(pI)is 8.40 and 8.82,all of which are unstable proteins.Analysis of the transmembrane region,hydrophobicity and signal peptide of Cry1 and Cry2 proteins showed that the overall hydrophobicity was not strong and was a hydrophilic protein,no signal peptide,and not a secreted protein,which was consistent with its transcription inhibitor.Functional domain predictions indicate that both contain two highly conserved functional domains:DNA photolyase and FAD binding domains.The analysis of phylogenetic relationship showed the C.barabensis has a close relationship with the C.griseus,while it is far from the primate and the chicken.These results consistent with the traditional classification of species,which suggesting the conservation of Cry1 and Cry2.This study plays a very important role in exploring the role of Cry1 and Cry2 about animal reproduction.