独脚金AFLP反应体系的优化及引物筛选  

Optimization of AFLP Reaction Systems and Primers Screening for Striga asiatica

作　　者：胡小虎 卢恩科 包日镇 陈泓烨 陈玫汛 黄光哲 林晋南 陈柯欣 梁丽娜 蔡玉冰 郭艺鹏 Hu Xiaohu;Lu Enke;Bao Rizhen;Chen Hongye;Chen Meixun;Huang Guangzhe;Lin Jinnan;Chen Kexin;Liang Lina;Cai Yubing;Guo Yipeng(Key Laboratory for Conservation and Utilization of Subtropical Bio-Resources,Education Department of Guangxi Zhuang Autonomous Region,College of Biology and Pharmaceuticals,Yulin Normal University,Yulin,537000)

机构地区：[1]玉林师范学院生物与制药学院,广西高校亚热带生物资源保护与利用重点实验室,玉林537000

出　　处：《分子植物育种》2023年第2期566-573,共8页Molecular Plant Breeding

基　　金：广西自然科学基金青年科学基金项目(2019GXNSFBA245038);广西科技基地和人才专项(桂科AD20159013);国家级大学生创新创业训练计划项目(202010606044);玉林师范学院博士科研启动项目(G2019ZK32)共同资助。

摘　　要：为了构建一套适合中国独脚金种质资源的AFLP反应体系,本研究以独脚金幼嫩茎叶为材料,对影响AFLP反应体系的酶切连接反应、预扩增和选择性扩增过程中的一些关键因素进行优化,并利用优化的反应体系对AFLP引物进行筛选。结果表明,最佳的酶切反应体系为DNA模板200 ng,MseⅠ/EcoRⅠ用量3 U,总体积20μL,37℃酶切反应时间3 h,72℃灭活20 min;最佳连接反应体系为T4连接酶5 U,16℃连接过夜;最佳预扩增反应体系为dNTP浓度0.2 mmol/L,Taq DNA聚合酶浓度2 U,总体积25μL;选择性扩增的反应体系为d NTP浓度0.2 mmol/L,Taq DNA聚合酶浓度1 U,预扩增产物稀释5~10倍,总体积10μL。采用优化的AFLP反应体系,对来自3个不同来源地的独脚金样本进行选择性扩增,100对引物组合均能扩增出清晰、丰富的条带,且在参试样本中均能表现出一定的扩增多态性。本研究筛选出的引物组合可用于后续独脚金种质资源遗传多样性的研究,为独脚金遗传多样性研究、种质资源保存和鉴定提供一定的参考。In order to construct a set of AFLP reaction system suitable for Striga asiatica germplasm resources in China,some key factors affecting digestion and linkage reaction,pre-amplification and selective amplification were optimized using young stems and leaves as experimental materials,and then primer combinations were screened.The results showed that,total 20μL of the optimal digestion reaction system containing 200 ng DNA template,3 U MseⅠand 3 U EcoRⅠ,was reacted at 37℃for 3 h,then inactivated at 72℃for 20 min;the optimal ligation reaction system containing 5 U T4 ligase was ligated overnight at 16℃;total 25μL of the optimal preamplification reaction system containing 0.2 mmol/L dNTP,2 U Taq DNA polymerase;total 10μL of the optimal selective amplification reaction system containing 0.2 mmol/L dNTP,1 U Taq DNA polymerase,1μL of 5 to 10-fold diluted pre-amplified products.All of the 100 pairs of selective-amplification primer combinations in the primer screening experiment could amplify clear and abundant bands using the optimal system,and exhibit a certain level of expanded polymorphism among samples from three different geographical origins.The primer combinations screened in this study can be used for the subsequent research on the genetic diversity of Striga asiatica germplasm resources,and provide the certain foundation for the research on genetic diversity,conservation and identification of Striga asiatica germplasm resources.

关 键 词：独脚金 AFLP 体系优化 引物筛选 

分 类 号：S567.239[农业科学—中草药栽培]