香石竹DcPAL1基因的克隆及其原核表达

Cloning and Prokaryotic Expression of Carnation DcPAL1 Gene

苯丙氨酸解氨酶(PAL)是植物苯丙烷类代谢途径的关键酶。利用RT-PCR及RACE技术从香石竹茎中克隆到苯丙氨酸解氨酶基因的全长cDNA,命名为DcPAL1(GenBank登录号为FJ864719)。DcPAL1全长2 397bp,预测其开放阅读框长2 121bp,编码一个含有706个氨基酸的蛋白质,其分子量为76.8kD,等电点5.84,且含有PAL的特征序列和活性位点。构建pET-DcPAL1原核表达载体,并转入大肠杆菌(Escherichia coli)中表达,获得1个与预测大小一致的外源蛋白,主要以包涵体形式存在。用Ni2+-NTA螯合琼脂糖层析柱亲和层析得到了纯化的表达蛋白。

Phenylalanine ammonia-lyase（PAL,EC 4.3.1.24） is the key enzyme of general phenylpropanoid metabolic pathway. A PAI. gene, designated as DcPAL1 （GenBank accession number： FJ864719）, was cloned from carnation （Dianthus caryophyllus cv. Master） stems. DcPAL1 has a length of 2 397 bp with an open reading frame of 2 121 bp encoding a protein of 706 amino acids. A typical sequence of PAL active region and other active sites can be found in the deduced protein. The molecule mass of DcPAL1 was 76.8 kD with estimated pI of 5.84. The expression vector pET-DcPAL1 was constructed and expressed in Esch- erichia coll. The expressed DcPAL1 protein exists in the form of inclusion body. The purified protein was obtained by affinity chromatography using Ni-NTA chelating agarose column.