摘要
以闹羊花的叶片为试材,采用正交实验和单因素试验2种方法对闹羊花ISSR-PCR反应体系的主要影响因子(Taq DNA聚合酶、dNTPs、模板DNA、引物、Mg2+以及反应程序)进行了优化组合。以期为深入开展闹羊花种质资源遗传多样性的研究提供参考依据。结果表明:闹羊花ISSR-PCR反应体系中各组分的Taq DNA聚合酶用量为2.5 U,引物浓度为0.2μmol·L-1,Mg2+浓度为1.0 mmol·L-1,dNTPs浓度为0.4 mmol·L-1,模板DNA浓度为60 ng,10×Buffer为2μL,以纯水补足反应总体积至20μL。扩增程序为94℃条件下预变性5 min;94℃条件下变性30 s,49.6℃条件下退火30 s,72℃条件下延伸30 s,以上3个步骤循环45次,最后72℃延伸5 min。
The leaves of Rhododendronmolle were used as the test materials.Through orthogonal design and single factor test,to establish ISSR-PCR program for Rhododendron molle.Five factors were tested by orthogonal design and single factor test,Mg2+ concentration,dNTP concentration,Taq DNA polymerase concentration,primer concentration,template DNA concentration and reaction procedure.The results showed that,the Taq DNA polymerase amount was 2.5 U,the primer concentration was 0.2μmol·L-1,the Mg2+ concentration was 1.0 mmol·L-1,the dNTPs concentration was 0.4 mmol·L-1,and the DNA concentration was 60 ng.Amplification procedure:pre-denaturation for 5 minutes at 94 ℃;denaturation for 30 seconds at 94 ℃,annealing for 30 seconds at 49.6 ℃,extension for 30 seconds at 72 ℃,45 cycles of the above three steps,and extension for 5 minutes at72℃.The establishment of this optimization system could be helpful for further research on the genetic diversity of germplasm resources of Rhododendron molle.
作者
覃芳
史艳财
邹蓉
唐健民
蒋运生
熊忠臣
QIN Fang;SHI Yancai;ZOU Rong;TANG Jianmin;JIANG Yunsheng;XIONG Zhongchen(College of Science,Guangxi Normal University,Guilin,Guangxi 541006;Guangxi Institute of Botany,Guangxi Zhuangzu Autonomous Region and the Chinese Academy of Sciences,Guilin,Guangxi 541006)
出处
《北方园艺》
CAS
北大核心
2020年第21期96-102,共7页
Northern Horticulture
基金
2019年广东省科技专项资金资助项目(2019B020201)
广西植物研究所基本业务费资助项目(19004)。
