苜蓿叶绿体DNA的提取及其Rbcl基因片段的克隆  

Extraction of Alfalfa Chloroplast DNA and Cloning of Rbcl Gene Fragment

作　　者：朱海林[1,2] 刘秀明[1,2] 江莺[1,3] 杜美丽[1,2] 李巍[1,2] 李海燕[1,2] 李校堃[1] 

机构地区：[1]吉林农业大学生物反应器与药物开发教育部工程研究中心,吉林长春130118 [2]吉林农业大学生命科学学院,吉林长春130118 [3]吉林农业大学中药材学院,吉林长春130118

出　　处：《安徽农业科学》2011年第22期13306-13308,共3页Journal of Anhui Agricultural Sciences

基　　金：国家"863"高新技术研究与发展计划项目生物反应器重大专项计划项目(2007AA100503);吉林省科技发展计划重点项目(20070922);吉林省科技厅"利用紫花苜蓿作为反应器生产抗菌肽的研究"(20100147);高等学校科技创新工程重大项目培育资金项目(70S018);吉林农业大学校内启动基金"抗菌肽转基因紫花苜蓿的表达研究"(201032)

摘　　要：[目的]获得高纯度的苜蓿叶绿体DNA(cpDNA)和Rbcl基因片段。[方法]采用改进的高盐低pH法分离和纯化苜蓿叶绿体DNA;利用叶绿体基因组进化中高度保守的特点,设计引物,从苜蓿叶绿体基因组中扩增Rbcl基因,利用生物学软件对测序结果进行分析。[结果]经检测分析,获得了产率较高和纯度较好的苜蓿叶绿体DNA,并用PCR方法扩增获得了1 130 bp的Rbcl基因片段。该基因片段有17个常用的限制性内切酶酶切位点,与烟草、水稻、菜豆、玉米、甘薯和甜菜中Rbcl基因核苷酸的同源性为85.6%～90.6%。[结论]为构建苜蓿叶绿体表达载体和通过叶绿体生物反应器生产药用蛋白等奠定了基础。[Objective] The aim was to obtain high purity alfalfa chloroplast DNA and Rbcl gene fragments.[Method] The improved high-salt and low pH method was adopted to isolate and purify chloroplast DNA（cpDNA）from alfalfa;based on the highly conserved features in chloroplast genome evolution,primers were designed and Rbcl gene was amplified from the alfalfa chloroplast genome,the results of sequencing by biological software were analyzed.[Result] Alfalfa chloroplast DNA with higher yield and precise purity were obtained by the detection and analysis.Also,1 130 bp of Rbcl homologous gene were obtained by PCR.The gene fragment has 17 common constrained incision enzyme restriction cutting site,the homologous of Rbcl with tobacco,paddy rice,kidney bean,maize,sweet potato and beet was 85.6%-90.6%.[Conclusion] The study will lay a foundation for constructing expression vectors and producing pharmaceutical protein through alfalfa chloroplast bioreactor.

关 键 词：苜蓿 叶绿体DNA 提取 RBCL基因 克隆 

分 类 号：S188[农业科学—农业基础科学]