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TAIL-PCR方法快速克隆银杏查尔酮合成酶基因及序列分析(英文)
引用本文:许锋,程水源,王燕,李琳玲,程述汉.TAIL-PCR方法快速克隆银杏查尔酮合成酶基因及序列分析(英文)[J].果树学报,2007,24(2):237-243.
作者姓名:许锋  程水源  王燕  李琳玲  程述汉
作者单位:1. 长江大学园艺园林学院,湖北荆州,434025;山东农业大学园艺科学与工程学院,山东泰安,271018
2. 长江大学园艺园林学院,湖北荆州,434025;黄冈师范学院生命科学与工程学院,湖北黄冈,438000
3. 长江大学园艺园林学院,湖北荆州,434025
4. 山东农业大学园艺科学与工程学院,山东泰安,271018
基金项目:教育部跨世纪优秀人才培养计划 , 教育部地方重点攻关项目 , 湖北省自然科学基金 , 湖北省杰出青年科学基金 , 湖北省教育厅科技攻关项目
摘    要:热不对称交错PCR(TAIL-PCR)方法已广泛应用于从多种生物克隆已知DNA序列的侧翼序列。对传统的TAIL-PCR方法进行改良:(1)将TAIL技术应用于TAIL-PCR中第3步PCR循环。(2)以10bp的随机简并引物即RAPD引物代替基因侧翼简并引物。以银杏品种家佛手的叶基因组DNA为模板,利用简并引物克隆到银杏查尔酮合成酶基因(CHS)片段序列Gbchs1,以此序列设计3条特异引物,利用改良的热不对称交错PCR方法克隆到CHS基因Gbchs2。结果表明,Gbchs2长1238bp,编码304个氨基酸并包含3’末端序列。GbCHS2蛋白质序列与其他植物的CHS蛋白质序列高度同源,包含CHS蛋白质保守的环化作用活性位点,催化活性位点、香豆素活性位点、及催化活性基序。改良后的TAIL-PCR方法为基因全长的克隆提供了一种简单快速高效的新方法。

关 键 词:银杏  热不对称交错PCR  查尔酮合成酶基因  克隆  序列分析
文章编号:1009-9980(2007)02-237-07
修稿时间:2006-08-25

Efficient amplification and sequence analysis of chalcone synthase gene from Ginkgo biloba by thermal asymmetric interlaced PCR
XU Feng,CHENG Shui-yuan,WANG Yan,LI Lin-ling,CHENG Shu-han.Efficient amplification and sequence analysis of chalcone synthase gene from Ginkgo biloba by thermal asymmetric interlaced PCR[J].Journal of Fruit Science,2007,24(2):237-243.
Authors:XU Feng  CHENG Shui-yuan  WANG Yan  LI Lin-ling  CHENG Shu-han
Institution:1.College of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei 434025 China; 2.College of Horticulture Science and Engineering, Shandong A gricultural University, Taian , Shandong 271018 China; 3.College of Life Science and Engineering, Hnanggang Normal University, Huanggang,Hubei 438000 China
Abstract:TAIL-PCR is a powerful tool for the recovery of DNA fragment adjacent to known sequences.With a modified TAIL-PCR technique,the genomic DNA sequence(named as Gbchs2)of chalcone synthase gene fragment was cloned from Ginkgo biloba cv.Jiafoshou.Two novel modifications in the TAIL-PCR procedures were introduced here:(1)the use of TAIL-PCR cycle in tertiary PCR,and(2)the use of a battery of random 10 bp RAPD primers as the short arbitrary primers.Gbchs2 was 1 238 bp long,containing 3'-flanking regions and encoding 304 amino acids.GbCHS2 was found to have extensive homology with those of other plant CHS based on multiple alignments,and the active site of the CoA binding,cumaroyl pocket,cyclization pocket and active site motif in CHS protein of other plants were also found in GbCHS2.The results showed that TAIL-PCR was useful,simple and powerful method for cloning full-length sequence of genes.
Keywords:Ginkgo biloba  TAIL-PCR  Chalcone synthase gene  Cloning  Sequence analysis
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