沙子岭猪跨膜与卷曲螺旋域2(TMCO2)基因克隆、生物信息学及组织差异表达分析

Cloning,Bioinfornatics and Tissue Differential Expression Analysis of Pig Transmenbrane and Coiled-coil Domains 2(TMCO2)

利用RACE法克隆沙子岭猪TMCO2基因cDNA全长并进行生物信息学分析,应用实时荧光定量PCR(qRT-PCR)对TMCO2基因在沙子岭猪D30时期8个不同组织(睾丸、肌肉、肺、心脏、肾脏、肝脏、脾脏、脂肪)及睾丸组织8个不同发育阶段(E90、D1、D30、D60、D90、D120、D150、D180)进行差异表达分析。结果表明:TMCO2基因克隆所得片段长740 bp,其中ORF区为546 bp,编码181个氨基酸。qRT-PCR结果显示,沙子岭猪D30时期TMCO2 mRNA在睾丸组织中表达量最高,其次为脾脏组织,而在其他组织中表达量极低,其中睾丸组织与其他组织表达量差异均为显著(P<0.05);睾丸组织不同发育时期中,TMCO2 mRNA在D180时期表达量达到最高,在D1和D150时期表达量略有下降,其中D180与其他时期的差异均为极显著(P<0.01),E90、D1、D30、D60 4个生长发育期时期表达量差异不显著(P>0.05)。本试验首次克隆了沙子岭猪TMCO2基因cDNA全长并检测了其在猪不同组织及睾丸组织不同发育阶段表达量,为进一步研究TMCO2基因功能奠定基础。

In this study, the TMC02 cDNA full length of pig was cloned by RACE and analyzed by bioinformaties, the TMC02 gene expression levels were detected in Shaziling pig D30 period of eight different tissues （testis, muscle, lung, heart, kidney, liver, spleen, fat） and eight different developmental stages （E90, D1, D30, D60, D90, D120, D150, D180） of testicular tissue by real-time fluorescence quantitative PCR. The results showed that the length of TMC02 eDNA and the ORF region were 740 bp and 546 bp, respectively, which encoded 181 amino acids, qRT-PCR results showed that the D30 period＇s highest expression level of TMC02 mRNA was found in testis, followed by spleen tissues, while the expression level of the other tissues were extremely low, the expression levels between testis and other tissues differed significantly （P〈0.05）. In the testis tissue, the expression level of TMC02 mRNA was the highest in D180 periods, while declined in DI and D150, the differences between D180 and the other periods were significant （P〈0.01）. The growing and development stages of E90, D1, D30 and D60 had no significant differences （P〉0.05）. The full length of TMC02 cDNA was cloned and the expression levels of TMC02 gene in different tissues and different developmental stages of testes were detected in this study, which provided the foundation for further understanding functions of the TMC02 gene in pig.