丰水梨多酚氧化酶基因的克隆与原核表达

The Cloning and Prokaryotic Expression of Polyphenol Oxidase Gene in Pear(Pyrus Pyrifolia Nakai)

通过PCR方法克隆丰水梨PPO基因全长,并将其登录Genebank,登录号JQ861265.基因全长1782 bp,无内含子,编码的PPO属于亲水性蛋白质,无跨膜结构,含有593个氨基酸,分子量约为65.8 kDa,理论等电点为8.4.N端含有一段由47个氨基酸组成的转运肽.去除转运肽的成熟PPO分子量为60.8 kDa,理论等电点为6.69.PPO中含有两个铜离子结合区,主要位于PPO二级结构中的α-螺旋区域中.原核诱导目的蛋白在诱导3~6 h后积累量较大.诱导蛋白（PPO前体和成熟PPO）均能氧化儿茶素形成茶黄素.

The polyphenol oxidase （PPO, GenBank accession No. JQ861265） genes were cloned by PCR from Pyrus Pyrifolia Nakai. The full length of PPO gene was 1 782 bp without introns, coding a precursor peptide. PPO precursor consists of 593 amino acids with a molecular weight of about 65.8 kDa. It has a theoretical PI of 8.4 and has a transit peptide consisting of 47 amino acids. The mature PPO without transit peptide consists of 547 amino acids with a molecular weight of about 60.8 kDa and a theoretical PI of 6.69. There are 2 Cu-binding domain in the α-helix of PPOs. The protein accumulation got a peak in 3-6 hours. The induced proteins （precursor PPO and mature PPO） can oxidize catechins into theaflavins in vitro.