珊瑚菜ITS序列的PCR扩增与测序分析

PCR Amplication and Sequencing Analysis of Glehnia Littoralis with ITS Fragments

为加强珍稀濒危物种珊瑚菜遗传多样性的研究和种质资源的保护工作,本实验对珊瑚菜ITS基因片段的PCR扩增条件进行了优化和产物的测序分析.结果显示:珊瑚菜ITS基因片段的PCR扩增最佳反应体系为Buffer 2. 50μL,d NTP 0. 50μL,引物ITS1和ITS4各1. 00μL,Taq DNA polymerase 0. 15μL,模板DNA 2. 00μL,dd H2O 17. 85μL,总体积25. 00μL.最佳扩增程序为94℃预变性5. 0 min,94℃变性50 s,50℃退火50 s,72℃延伸1 min 45 s,37个循环,后72℃延伸10 min.在最佳扩增条件下获得了清晰、稳定的目的条带,测序后获得长度为628 bp的核苷酸序列,通过GenBank的BLAST比对验证,确定为ITS序列,其中包括完整的ITS1序列和5. 8S r DNA序列以及ITS2的部分序列.

In order to strengthen the research of genetic diversity and the protection of germplasm resourses for the rare and endangered Glehnia littoralis ,we explored the optimized conditions for PCR amplification of the ITS gene and carried out sequencing analysis.The results showed that the optimized PCR amplification condition of PCR system was as follows：PCR Buffer 2.50 μL,dNTP mixture 0.50 μL,ITS1 1.00 μL,ITS4 1.00 μL,Taq DNA polymerase 0.15 μL,DNA 2.00 μL,ddH 2O 17.85 μL,the total volume was 25.00 μL.The optimized PCR amplification program was as follows：initial template denaturated at 94 ℃ for 5 min,94 ℃ for 50 s,50 ℃ for 45 s,and 72 ℃ for 1 min 45 s,followed by 37 cycles,with a final extension of 72 ℃ for 8 min and save at 4 ℃.A clear and definite target band was obtained under the optimal amplification condition.After sorting by hand,the total alignment of ITS sequences was 628 bp in length.The ITS gene was determined by the BLAST of GenBank,including the complete sequence of ITS1,the total sequence of 5.8S rDNA and the partial sequence of ITS2.