[铁皮石斛蔗糖合酶基因的分子克隆与表达分析]

[Molecular cloning and expression analysis of sucrose synthase gene from Dendrobium officinale].

作者信息

Meng Hengling, Duan Chengli, Xiao Fenghui, Yang Shengchao, Zha Yinghong, Wen Guosong

机构信息

Institute of Chinese Medical Materials, Yunnan Agricultural University/Yunnan Provincial Good Agricultural Practice Center of Chinese Medical Materials, Kunming 650201, China.

出版信息

Zhongguo Zhong Yao Za Zhi. 2011 Apr;36(7):833-7.

PMID:21761718

Abstract

OBJECTIVE

Clone of sucrose synthase of Dendribium officinale and expression analysis, to provide the theory basis for research the relationship between polysaccharide synthesis of D. officinale and sucrose synthase activity.

METHOD

According to homologous sequence of sucrose synthase gene on GenBank, application the technology of RT-PCR and RACE, clone the full length of D. officinale. Target gene amplified with T vector was transformed into competent E. coli. BL21, IPTG induced expression, SDS-PAGE analysis.

RESULT

A full length cDNA encoding sucrose synthase was isolated from the D. officinale, named DOSS1, the GenBank accession number is HQ856835, the cDNA is 2781 bp in length containing an open reading frame of 2424 bp encoding 807 amino acids with a predicted molecular mass of 92.3 x 10(3), the deduced amino acid sequence of D. officinale sucrose synthase shares 95% identity with Mokara yellow (AF530568); shares 90% identity with Oncidium goldiana (AF530567); shares more than 80% with other monocotyledonous plants.

CONCLUSION

Cloned the sucrose synthase gene and induced an obvious band successfully.

摘要

目的

克隆铁皮石斛蔗糖合成酶基因并进行表达分析，为研究铁皮石斛多糖合成与蔗糖合成酶活性之间的关系提供理论依据。

方法

根据GenBank上蔗糖合成酶基因的同源序列，应用RT-PCR和RACE技术克隆铁皮石斛蔗糖合成酶基因全长。将连接到T载体上的目的基因转化到大肠杆菌BL21感受态细胞中，经IPTG诱导表达后进行SDS-PAGE分析。

结果

从铁皮石斛中分离得到一个编码蔗糖合成酶的全长cDNA，命名为DOSS1，GenBank登录号为HQ856835，该cDNA全长2781 bp，包含一个2424 bp的开放阅读框，编码807个氨基酸，预测分子量为92.3×10³，铁皮石斛蔗糖合成酶推导的氨基酸序列与莫氏黄（AF530568）的一致性为95%；与文心兰（AF530567）的一致性为90%；与其他单子叶植物的一致性超过80%。