版纳微型猪近交系TDRP1基因克隆、鉴定及mRNA的组织表达特性

目的 克隆版纳微型猪近交系(BMI)不育和可育公猪TDRP1基因,分析其序列及mRNA表达水平上的差异,预测其蛋白质功能,并检测该基因在可育公猪中的组织表达分布情况.方法 以猪NM_001198925序列为模板,设计特异引物,采用RT-PCR方法结合测序获得TDRP1的cDNA序列并进行生物信息学分析;采用半定量PCR方法检测TDRP1在不育和可育公猪睾丸中的表达规律,分析该基因在可育公猪17种组织中的表达特征.结果 获得了BMI TDRP1基因的编码区序列(GenBank登录号:KJ186786),生物信息学分析表明其编码186个氨基酸,蛋白质相对分子质量(Mw)为20.49 ×10³,等电点(pI)为5.86,无信号肽,有94.1%的概率位于细胞核,含有1个亮氨酸富集的核输出信号.不同物种的氨基酸序列比对表明猪TDRP1与人、恒河猴、小鼠和大鼠等哺乳动物的TDRP1相似性在73%～83.2%之间,其中与人、恒河猴的相似性较高.mRNA表达分析表明,TDRP1在BMI不育和可育公猪睾丸间表达水平差异无显著,在精囊腺和前列腺中高表达,在睾丸和小脑中中度表达,在大脑和肾脏中低表达,在其余组织中不表达.结论 成功克隆了BMI TDRP1基因的全长编码区序列并发现了BMI特有的2个SNP位点;TDRP1基因在BMI不育和可育公猪间序列完全一致,睾丸mRNA表达水平差异无显著性,多组织转录谱分析表明该基因存在明显的组织差异表达现象,在精囊腺和前列腺中有较高表达量,为深入研究TDRP1基因在精子发生方面的作用奠定了基础.

Molecular cloning, sequence characterization and mRNA tissue expression analysis of TDRP1 gene from the Banna minipig inbred line (BMI)

Objective To get TDRP1 gene of sterile and fertile boar of the Banna minipig inbred line (BMI), predict its function by bioinformatics analysis, and detect its expression patterns in the fertile boar. Methods Based on the NM_001198925 sequence, we designed specific primers and amplified BMI TDRP1 using RT-PCR method for sequencing and bioinformatics analysis. Meanwhile, the expression of TDRP1 in 17 organ tissues (heart, liver, spleen, lung, kidney, thymus, lymph nodes, skin, duodenum, stomach, cerebrum, cerebellum,testis, epididymis, seminal vesicle, prostate, and bulbourethral gland) of fertile BMI boar and in the testis of sterile and fertile BMI boars was analyzed by semi-quantitative RT-PCR. Results The experiment obtained 680 bp cDNA sequence (GenBank accession number: KJ186786) of BMI TDRP1, which encodes a protein of 186 amino acids with a predicted molecular weight (Mw) of 20.49 kDa and isoelectric point (pI) 5.86, and no signal peptide. It was a nuclear protein with a probability of 94.1% and had a leucine-rich nuclear export signals. Homology analysis of protein sequences revealed that BMI TDRP1 showed high identity with that of humans, macaca mulatta, mouse and rat. The RT-PCR analysis showed that TDRP1 had a similar expression in the testes of sterile and fertile BMI boars. It was highly abundant in the seminal vesicle and prostate, moderately expressed in cerebellum and testis and weakly expressed in cerebrum and kidney, while undetected in other 11 organ tissues. Conclusions We have cloned TDRP1 complete coding sequence, and found 2 SNPs,showing no difference in sequences and the testis mRNA expression levels between the fertile and sterile BMI boars. The multi-tissue transcription profile shows different expression levels in different organ tissues, being high in the seminal vesicle and prostate. The results of this study provide a foundation for further insight into the role of this gene in spermatogenesis.