重叠延伸PCR法合成栀子两个糖基转移酶基因  

Synthesis of two glycosyltransferase genes involved in crocin biosynthesis in Gardenia jasminoides with PCR directed assembly from overlapping oligos

作　　者：王晓云[1,2,3] 雷志强[1] 王邵华[1] 王鹏良[4] 

机构地区：[1]江西中医药大学,江西南昌330004 [2]江西民族传统药现代科技与产业发展协同创新中心,江西南昌330004 [3]江西省中药种质资源工程技术研究中心,江西南昌330004 [4]广西钦州学院海洋学院,广西钦州535000

出　　处：《生物技术》2015年第5期451-457,462,共8页Biotechnology

基　　金：江西省卫生厅中医药科研基金课题("栀子糖基转移酶基因的克隆与功能分析";No.2012A142);国家自然科学基金项目("栀子果实中西红花总苷合成途径CCD基因的筛选与功能分析";No.31360362);江西省自然科学基金项目("栀子玉米黄素剪切加双氧酶基因的克隆与功能分析";No.20122BAB205070)资助~~

摘　　要：[目的]糖基转移酶UGT94E5和UGT75L6催化栀子果实中西红花总苷生物合成途径的末端步骤。该研究利用重叠延伸PCR法,合成编码这两个酶的基因序列。[方法]首先将基因分段,每段两端均加上p UC57载体的多克隆位点作为接头,拼接成800 bp以下的片段,再输入到DNAWorks软件中,获得最优寡核苷酸片段组合;将合成的寡核苷酸片段混合,用作模板,进行两次PCR扩增,获得加了接头的各段序列,亚克隆到自制的p UC57 T载体上。酶切后回收插入片段,混合,用作模板,扩增全长基因,产物克隆到自制的p UC57 T载体上。[结果]成功合成了栀子Gj UGT9(编码UGT94E5)和Gj UGT1(编码UGT75L6)基因,分别长1 496 bp和1 583 bp。[结论]重叠延伸PCR法能够有效地合成栀子两个糖基转移酶基因序列,为遗传操作奠定了基础。[ Objective] UGT94E5 and UGT75L6 are apocarotenoid glucosyhransferases that sequentially mediate the final glu- eosylation steps in eroein biosynthesis pathway in fruits of Gardenia jasminoides. In the present investigation, we used restriction endonuclease and PCR - directed gene synthesis from a group of overlapping oligodeoxyribonucleotides to obtain the two genes, i. e. GjUGT9 encoding UGT94E5 and GjUGT1 encoding UGT75L6, [ Methods] Each gene was divided into several sections flan- king with restriction endonuclease recognition sites and vector fragments for directional cloning. These sections were assembled into sequences less than 800bp, and input to DNAWorks. A series of oligonucleotide sequences were produced, optimized, chem- ically synthesized,combined and assembled in a two - step PCR protocol to form the synthetic sections. These synthetic sec- tions, purified by gel extraction, were inserted into pUC57 vector after enzyme digestion, and then transformed into E. coli cells. Those plasmids containing correct target fragments were identified, sequenced, and digested by restriction endonucleases. The yielded target fragments were recovered, mixed and used as template for next round amplification of the full length of GjUGT9 or GjUGT1. Then the PCR products were ligased into pUC57 - T vector through TA cloning. Those constructed plasmids were screened by restriction endonuelease digestion, and submitted for sequencing. [ Results ] We have successfully obtained the 1496 bp GjUGT9 gene and 1 583 bp GjUGT1 gene with this synthetic method. [ Conclusion] The restriction endonuclease and PCR directed DNA synthesis protocol described in this paper is effective for gene synthesis.

关 键 词：基因 合成 栀子 糖基转移酶 重叠延伸PCR法 

分 类 号：Q523[生物学—生物化学]