斑节对虾腺苷酸转移酶(PmANT)基因的cDNA克隆与表达分析

Molecular cloning and expression analysis of adenine nucleotide translocase(PmANT) in Penaeus monodon

利用RACE技术获得了斑节对虾(Penaeus monodon)ANT基因(PmANT)的cDNA序列。该序列全长1 388 bp,开放阅读框(ORF)为930 bp,3'非编码区(UTR)为393 bp,5'非编码区(UTR)为65 bp。ORF可编码309个氨基酸,分子量大约为33.622 ku。与所有ANT家族成员一样,PmANT蛋白具有3个重复同源的线粒体跨膜结构域,但不含信号肽和糖基化位点。相似性、同源性及系统进化树分析显示,斑节对虾的ANT基因与凡纳滨对虾的同源性和相似性最高,并与其聚为一支。采用荧光定量的方法研究了ANT基因在雌雄个体不同组织、卵巢不同发育阶段及未成熟和成熟精巢的差异表达情况。结果表明:PmANT的mRNA在各组织中都有表达,其中,在雄性个体的肌肉中表达量最高,其次为雌性肌肉,在精巢的表达量最低,且未成熟精巢低于成熟精巢。PmANT的mRNA在卵巢的表达量高于精巢,且在Ⅲ期卵巢表达量最高,Ⅳ期最低。为今后进一步研究该基因在斑节对虾性腺发育中的作用提供基础材料。

The adenine nucleotide translocase（ANT） is the most abundant mitochondrial inner membrane protein,which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis.In the present study,the full sequence of P.monodon ANT gene was cloned and named PmANT.The full length cDNA of PmANT contained a 5′ untranslated region（UTR） of 65 bp,a 3′ UTR of 393 bp and an ORF of 930 bp encoding a polypeptide of 309 amino acids with an estimated molecular mass of 33.622 ku.Like other animal ANTs,the structure of the PmANT protein consists of three homologous repeated domains.But there are no signal peptide and glycosylation sites in PmANT protein.Sequence alignment analysis showed that the PmANT with the ANT of Litopenaeus vannamei shared a similarity of 98.7% and the homology of 97.4%.Analysis of the tissue expression pattern of the PmANT showed that the PmANT mRNA was expressed in all tested tissues,including heart,ovary,testis,muscle,eye stable nerve,cranial nerve,lymphoid tissue,stomach,intestines,hepatopancreas,gill,hemocyte,with the highest levels in muscle of male and the lowest level in immature testis.Furthermore,the PmANT expression was found to be at a higher level in mature testis than in immature testis.The PmANT expression was found in the six ovarian stages of development and the expression level of ovarian growth in the third growth phase was significantly higher than that of others except the sixth growth phase,while lowest in the fourth growth phase.The study provided essential materials and laid a firm foundation for the further research of the function of PmANT in gonad development of P.monodon.