欧李PSY基因cDNA克隆与表达分析.doc

文档介绍：
欧李 PSY 基因 cDNA 克隆与表达分析#
张建成,王鹏飞,刘和,薛晓芳,穆霄鹏,杜俊杰**
(山西农业大学园艺学院,山西太谷 030801)
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摘要:利用 GenBank 登录的植物八氢番茄红素合成酶(phytoene synthase,PSY)氨基酸保
守区序列设计简并引物,通过 RT-PCR 和 RACE-PCR 技术从欧李果实中获得了一个 PSY
cDNA 全长序列,命名为 ChPSY。序列分析表明,该基因 cDNA 全长 1 559 bp,最大开放读
码框为 1 194 bp,编码 397 个氨基酸,与草莓 PSY 氨基酸序列的同源性为 %,与辣椒、
番茄、拟南芥、柑橘、甜瓜、番木瓜、玉米和水稻等其它植物的同源性为 %~%。
RT-PCR 分析表明,ChPSY 在欧李的花、叶片和果实中均有表达,表达量依次为花>深红果
>橙红果>泛白果>青绿果>老叶>幼叶。工程大肠杆菌异源表达体系证实 ChPSY 可编码一个
功能蛋白,催化工程菌株中 2 分子 GGPP 缩合生成八氢番茄红素。
关键词:欧李;八氢番茄红素合成酶;cDNA 克隆;表达分析
中图分类号:S
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Cloning and Expression Analysis of PSY Gene from Cerasus
humilis(Bge)Sok
ZHANG Jiancheng, WANG Pengfei, LIU He, XUE Xiaofang, MU Xiaopeng, DU
Junjie
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(College of Horticulture, Shanxi Agricultural University, Taigu Shanxi 030801)
Abstract: Using PCR degenerate primers designed based on the conserved amino acid sequences
of known plant PSY to amplify cDNA fragments from the fruit of Cerasus humilis by RT-PCR
and RACE-PCR, a PSY gene named ChPSY was cloned. Sequence analysis indicated that the
full-length cDNA was 1 559 bp, which contained an open reading frame of 1 194 bp and encoded
a protein of 397 amino acids residues. The ChPSY protein showed % homology to the PSY
from strawberry, and also %~% homology to the PSYs from other plants, such as pepper,
Arabidopsis thaliana, citrus, melo, papaya, maize and rice. The semi-quantitative RT-PCR
analysis revealed that expression of ChPSY could be detected in different tissues of Cerasus
humilis. The expression level in the flower was found the highest, while that in the deep-red fruits,
the reddish-orange fruits, the white fruis, the green fruits and old leaf run the second, and that in
the new leaves paratively lower. The heterogenous expression in E. coli system
confirmed that ChPSY could encode a functional phytoene synthase which could catalyze a
head-to-head condensation of two molecular of GGPP to form phytoene in an E. coli engineered
to produ