水牛Tbx3基因的克隆及表达分析

Cloning and Expression Analysis of Buffalo Tbx3 Gene

转录因子Tbx3(T-box3)在调控动物个体的正常发育中扮演重要角色。本研究克隆水牛(Bubalus bubalis)Tbx3基因,对其进行生物信息学分析,并检测其在水牛不同发育阶段早期胚胎、不同类型细胞以及不同组织的表达情况,初步探讨Tbx3在水牛胚胎发育过程中的作用。根据GenBank上已登录的牛(Bos taurus)、山羊(Capra hircus)等物种的Tbx3基因序列设计特异性引物,利用RT-PCR技术和T-A克隆技术克隆水牛Tbx3基因,利用实时荧光定量PCR(RT-qPCR)技术检测、分析水牛Tbx3基因的表达谱。结果显示,克隆获得的水牛Tbx3基因长度为3291 bp,包含了编码728个氨基酸的编码区及其5′端1 kb非编码区片段。水牛Tbx3基因与牛、绵羊(Ovis aries)、猪(Susscrofa domestica)、黑猩猩(Pan troglodytes)、小鼠(Mus musculu)Tbx3基因的相似性分别为97%、98%、90%、88%和85%,且在不同物种间高度保守,符合生物进化规律。蛋白质结构预测分析软件提示,Tbx3蛋白包含59.48%无规则卷曲、32.28%α-螺旋、8.24%伸展串,为亲水不稳定的碱性蛋白。RT-qPCR结果显示Tbx3在水牛早期胚胎的四细胞、八细胞期不表达,在二细胞期、桑椹胚期少量表达,在囊胚期表达量最高;在水牛颗粒细胞中不表达,在胎儿成纤维细胞、骨髓间充质干细胞中有少量表达,在类胚胎干细胞中表达量最高;Tbx3在水牛卵巢、心脏、睾丸、脑、胚胎生殖嵴中少量表达,脾脏、肝脏表达更高,肾脏、肌肉中表达量最高。本研究发现水牛Tbx3基因的表达具有时空特异性,提示其在水牛早期胚胎细胞谱系分化中起重要作用,但具体作用机制尚需进一步研究阐明。

The T-box transcription factor Tbx3(T-box3)plays crucial roles in regulating the normal development of animals.In order to explore the role of Tbx3 in the embryo development of buffalo(Bubalus bubalis),Tbx3 gene of buffalo was cloned and the sequence was systemically analyzed by bioinformatics techniques as well as the expression patterns in different development stage of early embryo,different types of cells and different organizations.The specific primers of Tbx3 gene were designed in this experiment,and the Tbx3 gene was cloned by RT-PCR and T-A cloning technique.The Tbx3 sequence was systemically analyzed by bioinformatics techniques including gene homology,nucleic acid phylogenetic tree production,protein physical properties and advanced structure.The Tbx3 gene expression profiles were analyzed by real-time fluorescent quantitative PCR(RT-qPCR).The results showed that the buffalo Tbx3 gene was 3291 bp in lenth,which included coding sequence(CDS)including 728 amino acids and 1 kb non-coding region at the 5′end.The similarity of buffalo Tbx3 gene with Bos taurus,Ovis aries,Susscrofa domestica,Pan troglodytes and Mus musculus were 97%,98%,90%,88%and 85%respectively.Furthermore,protein structure prediction showed that buffalo Tbx3 was hydrophilic unstable basic protein contained 59.48%random coils,32.28%α-helice,8.24%extended strands.RT-qPCR results showed Tbx3 was not expressed in four cell and eight cell stage of buffalo early embryos,lightly expressed in two cell stage and morula stage,and expressed in a higher level in blastocysts.Interestingly,Tbx3 was not expressed in granulosa cells,lightly expressed in fetal fibroblast cells and bone marrow mesenchymal stem cells,but significantly expressed in the buffalo embryonic-like stem cells.Moreover,Tbx3 expressed in all nine tissues,but the expression level was higher in buffalo kidney and muscle,while the expression level was lower in ovaries,heart,brain,testis,and gonadal ridges.This study showed that the expression of buffalo Tbx3 had spatial-temporal specificity,which indicated that Tbx3 was one of the critical regulators in embryonic cell lineage specification.The underly mechanism of Tbx3 in buffalo embryonic cell lineage specification still need to be clarified in future.