鸭梨多酚氧化酶基因CDS区的克隆及表达

以鸭梨(Pyrus bretschneideri Rehd.)果皮基因组DNA为模板,根据已经发表的多酚氧化酶(polyphenol oxidase,PPO)基因保守序列设计引物,利用PCR技术,克隆得到鸭梨多酚氧化酶编码区序列,将此核酸序列克隆到载体pGEM-T,酶切鉴定后测序,结果表明该段序列含有1782个核苷酸,编码593个氨基酸。对鸭梨多酚氧化酶基因片段的一致性分析和进化树分析表明,该片段与沙梨(Pyrus pyrifolia,AB056680)、苹果(Malus domestica,L29450)、李(Prunuss alicina,AY866432)以及杏(Prunus armeniaca,AF020786)的一致性分别为99%、96.3%、82%和51.4%,绘制的进化树和形态学分类地位相一致。将该片段连接到表达载体pET39b上,获得的重组子命名为pET39b-PPO,热激法转化表达受体大肠杆菌BL21(DE3)菌株,用IPTG进行诱导。SDS-PAGE分析表明,PPO基因在大肠杆菌中被诱导表达蛋白质相对分子质量约66ku,检测表明具有多酚氧化酶的活性。

Cloning and expression of polyphenol oxidase gene of CDS from Yali pear

The codon domain sequence of polyphenol oxidase (PPO) gene of Yali pear (Pyrus bretschneideri Rehd.) was amplified by PCR method according to the template of genomic DNA of Yali pear, whose primers were designed by the conserved domain of the published PPO gene sequence in other plants. The fragment of PPO gene was cloned to the vector pGEM-T, then sequenced. Sequence analysis showed that the CDS of PPO gene in Yali pear consisted of 1 782 nucleotides, encoding a predicted polypeptide of 593 amino acids. Alignment of nucleotide sequence and other PPO genes showed that the PPO gene of Pyrus bretschneideri Rehd. had an identity of 99%,96.3%,82%and51.4% to PPO gene from Pyrus pyrifolia, Malus domestica, Prunus salicina, and P. armeniaca, respectively. The phylogenetic tree was congruent with morphological status. The CDS domain of PPO gene was cloned into pET39b expression vector, the recombined plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. Results of SDS - PAGE showed that a 66 ku protein was achieved, which had PPO activity.