花生磷酸烯醇式丙酮酸羧化酶基因的克隆及反义表达载体的构建

磷酸烯醇式丙酮酸羧化酶(PEPCase)是控制植物籽粒中蛋白质/油脂含量比例的关键酶.本研究利用RT-PCR技术,克隆PEPCase基因的片段,并将克隆的PEPCase基因片段反向连接替代植物表达载体PBI121的GUS基因.从花生栽培品种荔蒲大花生基因组中克隆获得编码PEPCase的基因片段(886 bp),其核苷酸序列与已报道的花生(EU391629)、棉花(AY008939)、大豆(D10717)、拟南芥(AY210895)、豌豆(D64037)PEPCase基因对应部分的同源性分别为99.77%,84.37%,81.54%,81.25%,78.71%,说明我们得到PEPCase基因片段较准确.构建的反义表达载体中PEPCase基因由35S启动子所控制,将构建的反义表达载体命名为pBGPEP.为通过反义抑制技术提高花生含油量提供了基因及表达载体.

Cloning of Peanut Gene Fragment Encoding PEPCase and Construction of Its Plant Expression Vector

Phosphoenolpyruvate carboxylase (PEPCase) plays an important role in the control of the ratio of the pro-tein/lipid content in plant seed. A 886 bp DNA fragment which was amplified by RT-PCR using PEPCase gene special primer from peanut seed. The DNA sequence analysis indicated that the sequence of this fragment was shared 99.77 % , 84.37%, 81. 54% ,81.25% ,78. 71% homology with the corresponding sequence of the reported PEPCase gene in peanut,cotton,soybean,thale cress,pea.The 886 bp fragment digested by BamH I and Sac I and ligated into the GUS site of the pBI121 in the antisense orientation and constructed the antisence expression vector pBGPEP.