CUI Caiyun,YAN Guohong,JIANG Shan.The Construction of Physcomitrella patens C3H Gene Knock Out Vector and Optimum Condition of PCR for C3H1-nptⅡ-C3H2 Target Fragment[J].Northern Horticulture,2016,40(05):113-118.[doi:10.11937/bfyy.201605031]
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
- Title:
- The Construction of Physcomitrella patens C3H Gene Knock Out Vector and Optimum Condition of PCR for C3H1-nptⅡ-C3H2 Target Fragment
- 关键词:
- 小立碗藓C3H基因; 基因敲除; 载体构建; 退火温度
- Keywords:
- Physcomitrella patens C3H gene; gene knock out; vector construction; annealing temperature
- 文献标志码:
- A
- 摘要:
- 以小立碗藓作为试材，将总长为1 981 bp的小立碗藓C3H基因分为上游片段（1 000 bp），下游片段（981 bp），并以此设计引物，得到上游臂（C3H1，735 bp）、下游臂（C3H2，700 bp）。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0.20 μmol/L，退火温度为58.7℃,PCR产物特异性最高且产物浓度较高。
- Abstract:
- Taking Physcomitrella patens as experimental materials,the full gene sequence of C3H that was 1 981 bp in the Physcomitrella patens would be divided into upstream fragment (1 000 bp)，the downstream fragment(981 bp).Primers were designed with upstream and downstream fragment，respectively.The first half was named upstream arm (C3H1，735 bp) and the latter half was called downstream arm (C3H2，700 bp).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 C3H2 were inserted into the pTN182 vector and knockout vector(C3H1-pTN182-C3H2 plasmid) 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 0.20 μmol/L and annealing temperature was 58.7℃.
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备注/Memo
第一作者简介：崔彩云(1990-)，女，河北沧州人,硕士研究生，研究方向为资源植物工程学。E-mail:ccydywe@sohu.com.责任作者：姜山(1969-),男，贵州贵阳人，博士，教授，研究方向为植物病理学。E-mail:kyosan200312@hotmail.com.基金项目：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)。