小立碗藓C3H基因敲除载体的构建及C3H1-nptⅡ?C3H2目的片段最佳PCR条件筛选  

The Construction of Physcomitrella patens C3H Gene Knock Out Vector and Optimum Condition of PCR for C3H1-nptⅡ-C3H2 Target Fragment

作　　者：崔彩云[1] 晏国洪 姜山[1] 

机构地区：[1]贵州师范大学生命科学学院,贵州贵阳550001

出　　处：《北方园艺》2016年第5期113-118,共6页Northern Horticulture

基　　金：国家自然科学基金资助项目(30860158);贵州省自然科学基金资助项目(31260426)

摘　　要：以小立碗藓作为试材,将总长为1 981bp的小立碗藓C3H基因分为上游片段(1 000bp),下游片段(981bp),并以此设计引物,得到上游臂(C3H1,735bp)、下游臂(C3H2,700bp)。在C3H1引物设计时引入XhoI、EcoRV酶切位点到两端,C3H2设计时引入NdeI、BamHI酶切位点到两端。以小立碗藓总DNA为模板,通过PCR扩增得到C3H1和C3H2片段。将C3H1和C3H2插入到pTN182载体中,获得敲除载体(C3H1-pTN182-C3H2质粒),通过PCR获得C3H1-nptⅡ?C3H2目的片段,并且采用单因素方法研究引物浓度与退火温度对PCR产物浓度及特异性的影响。结果表明:引物浓度为0.20μmol/L,退火温度为58.7℃,PCR产物特异性最高且产物浓度较高。Taking Physcomitrella patens as experimental materials,the full gene sequence of C3 Hthat was 1 981 bp in the Physcomitrella patens would be divided into upstream fragment（1 000bp）,the downstream fragment（981bp）.Primers were designed with upstream and downstream fragment,respectively.The first half was named upstream arm（C3H1,735bp）and the latter half was called downstream arm（C3H2,700bp）.Using the total DNA of Physcomitrella patens to template and the designed primer of C3H1 and C3H2,amplified C3H1 which was adding restriction site was XhoI and EcoRV and C3H2 which was adding restriction site was NdeI and BamHI by PCR.The C3H1 and C3H2were inserted into the pTN182 vector and knockout vector（C3H1-pTN182-C3H2plasmid）was succeed.C3H1-nptII-C3H2 fragment was obtained by PCR,The research of the primer concentration and annealing temperature that had an effect on the concentration and specific of the PCR product was by the single factor method.The results showed that the specificity and concentration of PCR product was higher when primer concentration was 0.20μmol/L and annealing temperature was

关 键 词：小立碗藓C3 H基因 基因敲除 载体构建 退火温度 

分 类 号：S781.42[农业科学—木材科学与技术]