人受精促进肽受体(TCP11c)基因的克隆、表达及选择性剪接

目的 分离、克隆人受精促进肽受体TCP11基因1个新的转录本TCP11c的全长cDNA,研究其表达的阶段性、组织特异性及TCP11c基因3种转录本的剪接方式。方法 运用BLAST方法获得与TCP11a同源的ESTs,以逆转录的人睾丸cDNA为模板进行PCR扩增,将PCR扩增片段克隆入p哪M—TeMy载体并516序;采用BLAST、ClustalW及RT—PCR方法分析各转录本的基因组结构及剪接方式;RT—PCR方法分析其组织表达的特异性和阶段性。结果 获得1个新的全长cDNA,它编码440个氨基酸的蛋白质,与TCP11a、b相比,在基因组的5′—端存在复杂的外显子剪接现象。RT—PCR结果显示该转录本在正常睾丸中表达,而其他组织、无精症患者及胎儿睾丸组织中未见该基因表达。结论 从人睾丸组织中分离到人受精促进肤受体TCP11基因1个新的转录本TCP1c,结合mTcp-11的功能提示,TCP11基因a、b、c这3种转录本对精于发生和人受精过程可能起重要作用。

Cloning,expression, and alternative splicing of the novel isoform of hTCP11 gene

OBJECTIVE: To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing. METHODS: According to the sequence of human ESTs which are highly homologous to hTCP11a, primers for PCR were synthesized. Then, the amplified fragments were cloned and sequenced; some methods including BLAST, ClustalW and RT-PCR were used for genomic analysis, study of alternative splicing and gene expression among multiple tissues and different testis tissues. RESULTS: A novel isoform of hTCP11 gene was isolated. It encodes a 440 amino acid protein that is highly homologous to the mouse 566 amino acid protein which is important to sperm function because it encodes the receptor for fertilization promoting peptide (FPP). Among TCP11a, TCP11b and TCP11c, the complicated alternative splicing was found. RT-PCR analysis of RNA extracted from human tissues revealed that the gene is only expressed in fertile adult testes, but not in azoospermic patient testes, fetal testes or other human tissues. CONCLUSION: Our results along with the mouse Tcp-11 function suggest that the isoforms of TCP11 gene play important roles in sperm function and fertility.