文献类型：期刊文献

中文题名：利用ISSR-PCR研究麻花秦艽超低温再生植株遗传稳定性

英文题名：Study on Genetic Stablity of Cryopreservation Regeneration Plant of Gentiana straminea with ISSR-PCR

作者：高素芳[1];陈红刚[1];张延红[1];李慧林[1];晋玲[1]

第一作者：高素芳

机构：[1]甘肃中医学院,甘肃兰州730000

第一机构：甘肃中医药大学

年份：2015

卷号：38

期号：6

起止页码：1123

中文期刊名：中药材

外文期刊名：Journal of Chinese Medicinal Materials

收录：MEDLINE(收录号：26762049);CSTPCD;;Scopus;北大核心:【北大核心2014】；CSCD:【CSCD2015_2016】；PubMed;

基金：甘肃省自然科学基金计划项目(BH2012-076);甘肃省中医药管理局项目(GZK-2010-Z2)

语种：中文

中文关键词：麻花秦艽;超低温再生植株;正交试验;ISSR-PCR;遗传稳定性

外文关键词：Gentiana straminea Maxim. ; Cryopreservation regeneration plant ; Orthogonal experiment ; ISSR-PCR ; Genetic stability

摘要：目的:建立麻花秦艽的ISSR-PCR最佳反应体系,筛选适宜引物,对麻花秦艽超低温再生植株进行遗传稳定性研究。方法:试剂盒法提取麻花秦艽超低温再生植株DNA,正交设计法筛选最佳ISSR-PCR反应体系,分析其遗传稳定性。结果:建立了麻花秦艽的ISSR-PCR反应最佳体系(25μL):d NTPs 0.50μL、Mg2+1.00μL、10×PCR Buffer 2.00μL、引物0.60μL、Taq酶1.25μL、模板DNA 1.30μL、dd H2O 18.35μL。扩增程序:94℃预变性5min,94℃变性30 s,(根据不同引物的退火温度)复性1 min,72℃延伸1.5 min,共35个循环,循环结束后72℃延伸7 min,4℃保存。麻花秦艽超低温再生植株的变异率为1.05%。结论:超低温保存获得的麻花秦艽再生植株株间变异率较低,具有较高的遗传稳定性,可以作为麻花秦艽资源保护的一种可行方法。
Objective ： To establish ISSR-PCR system of cryopreservation regeneration plant of Gentiana straminea, and to select ap- propriate primers and analyze the genetic stability. Methods ： DNA was extracted by CTAB, the optimal ISSR-PCR system was established by orthogonal experiment, and genetic stability was analyzed. Results ： The optimal ISSR-PCR system （ 25 μL） was established ： dNTPs 0. 50 μL, Mg^2 ＋1.00 μL, 10 × PCR Buffer 2. 00 μL, primer 0. 60 μL, Taq DNA polymerase 1.25 μL, template DNA 1.30 μL, and ddH20 18. 35 μL. The amplification program was devised：94 ℃ for 5 min, denaturing at 94 ℃ for 30 s, annealing of 1 min due to dena- turing temperature of different primer, extension at 72 ℃ for 1.5 min,35 cycles,last extension at 72 ℃ for 7 min, conservation at 4℃. The DNA mutation rate of cryopreservation regeneration plant of Gentiana straminea was 1.05%. Conclusion ：The cryopreservation re- generation plant of Gentiana straminea retains very good genetic stability, there is little variation between each plant, so the cryopreserva- tion can be used as a feasible method for resource protection of Gentiana straminea.