马蔺V-ATPase c亚基基因家族的克隆及序列分析

Cloning and Sequence Analysis of Vacuolar-type H^+ -ATPase Subunit c Family from Iris lactea

植物V-ATPase在逆境条件下的应答功能对保证细胞的正常生命活动具有重要作用。本研究根据GenBank收录的外源V-ATPase c亚基,设计简并引物,以马蔺为材料,通过RT-PCR进行扩增,经测序该片段为471bp,包含V-ATPase基因家族的保守结构域,以该片段为靶序列进行5′-RACE延伸,将产物测序得到186bp的序列,将靶序列与5′-RACE产物进行拼接,根据拼接结果设计特异引物,克隆基因全长读码框。结果表明,得到马蔺VHA-c亚基家族的5个成员,片段全长632bp,包括137bp5′-UTR,开放读码框全长495bp,编码164个氨基酸,分子量约为16kDa,包含4个跨膜结构域。将该基因命名为IrlVHA-c1～c5。

The response function of plant V-ATPase to adversity plays an important part of ensuring the normal vital movement of cells.According to the exogenous V-ATP c subunit gene which exusts in GenBank,degenerate primers were designed.471 bp of a fragment was cloned in Iris lactea through RT-PCR.The results showed that there was a high homology between the nucleotide sequence and the derived amino acid sequence of the fragment.This amino acid sequence included conserved structural domain of the V-ATP family.After RACE,the full length gene of 632 bp was obtained,with the ORF of 495 bp coding 164 amine acid,and the molecular weight of about 16 kDa,containing 4 membrane spaning domain.The gene was named IrlVHA-c1~c5.