红景天属植物DALP反应体系的建立

Establishment of DALP reaction system for plants of Rhodiola L.

目的 利用直接扩增片段长度多态(DALP)这一新分子标记技术建立一套稳定的红景天属植物的DALP反应体系。方法 以红景天基因组DNA为模板,对DALP反应程序的一些重要参数进行摸索和优化试验。结果 建立了一套稳定性强、可靠性高的DALP应用反应体系;经过大量重复性实验,反应体系为20μL,Mg2+浓度为2.5 mmol/L,dNTPs浓度为1.25 mmol/L,模板DNA的量为60 ng,5 pmol/L选择性引物1μL,5 pmol/L反向引物3 μL,引物浓度比为1:3,Taq酶2 U。反应过程为:95℃预变性5 min、94℃变性30 s、50℃退火30 s、72℃延伸1 min;30个循环,再72℃延伸10 min,完成整个PCR反应。结论 该体系可有效地应用于红景天属药材的分类鉴定和地道性鉴定。

Objective Direct amplification of length polymorphism (DALP) as a new molecular marker was used to establish a set of stable DALP reaction system for the plants of Rhodiola L. Methods Some significant parameters of DALP reaction procedure were investigated and optimized by taking the DNA genome for the plants of Rhodiola L. as template. Results The reaction system was : 20 μL reaction system containing 2. 5 mmol/L Mg2+ , 1. 25 mmol/L dNTPs, 60 ng DNA template, 1 μL 5 pmol/L selective primer, 3 μL 5 pmol/L reverse primer, selective primer: reverse primer is 1 : 3, and 2 U Taq DNA polymerase. Amplification program is 95℃ pre-denatured for 5 min, 94℃ denatured for 30 s, 50℃ annealed for 30 s, 72℃ extending for 1 min; after 30 cycles, and then 72℃ extending again for 10 min to the end of PCR reaction. Conclusion This DALP reaction system is efficient to identify the species and local populations for the plants of Rhodiola L. repeatedly with the stronger stability and reliability.