Error-pronePCR致哈茨木霉几丁质酶突变的条件优化

摘要：运用Error—pronePCR对哈茨木霉几丁质酶基因（mi珥2）进行人工突变。在常规PcR基础上，通过提高体系中Mg2＋浓度，适当加入一定浓度Mn2＋，调节四种dNTP（dATP、dGTP、dCTP、dGTP）浓度，得到产生明显突变效果的试验条件。在Error—pronePCR中，使用5．0mmol·L-1·Mg2＋，0．5mmol·L。Mn2＋，0．04mmol·L。dATP和dGTP，1．5mmol·L。dTTP和dCTP进行扩增后，随机挑选10个克隆测序，累积13370个碱基，检测到57个突变，平均突变率为0．4％。为产生优良突变株、酶分子定向进化奠定基础。...

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Published in东北农业大学学报 Vol. 44; no. 10; pp. 134 - 138
Main Author 王傲雪 李微 陈秀玲 李景富
Format Journal Article
LanguageChinese
Published 2013
Subjects
Chit42
Error—prone
PCR
哈茨木霉几丁质酶
突变
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Summary:摘要：运用Error—pronePCR对哈茨木霉几丁质酶基因（mi珥2）进行人工突变。在常规PcR基础上，通过提高体系中Mg2＋浓度，适当加入一定浓度Mn2＋，调节四种dNTP（dATP、dGTP、dCTP、dGTP）浓度，得到产生明显突变效果的试验条件。在Error—pronePCR中，使用5．0mmol·L-1·Mg2＋，0．5mmol·L。Mn2＋，0．04mmol·L。dATP和dGTP，1．5mmol·L。dTTP和dCTP进行扩增后，随机挑选10个克隆测序，累积13370个碱基，检测到57个突变，平均突变率为0．4％。为产生优良突变株、酶分子定向进化奠定基础。
Bibliography:23-1391/S
In this study, the Trichoderma harzianum chitinase gene（Chit42） was mutated by using Error-prone PCR method. On the basis of conventional PCR, the optimal conditions of Error-prone PCR were investigated by improving Mg2＊ concentration in PCR system, adding appropriate concentration of Mn2＋, adjusting the concentrations of four kinds of dNTP （dATP, dGTP, dCTP, dGTP）. Error-prone PCR was executed by adding 5.0 mmol. L1 Mg2＋, 0.5 mmol. L-1 Mn2＋, 0.04 mmol. L-1 dATP and dGTP; 1.5 mmol-L-1 dTTP and dCTP for amplifying the chit42 gene. Total 10 clones were selected at random, the sequences results showed that 57 mutations occurred in 13 370 nucleotides, 0.4% mutation rate can be obtained by Error-prone PCR. This study laid a solid groundwork to produce mutant for the directed evolution of enzyme molecule.
Error-prone PCR; Trichoderma harzianum chitinase; Chit42; mutation
WANG Aoxue＇＇2, LI Wei＇, CHEN Xiuling1, LI Jingfu＇（l. School of Horticuture, Northeast Agricultural University, Harbin 150030, China; 2. Sch
ISSN:1005-9369