芸芥柱头总RNA提取方法及自交亲和性相关基因mRNA差异显示分析体系研究

Studies on Methods for Extracting Total RNA from Stigmas and mRNA Reaction System for Screening Genes Related to Self-compatibility in E.Sativa Mill.

以芸芥自交亲和系为材料,用改进异硫氰酸胍法提取芸芥柱头总RNA,所提取的RNA完整性好、没有DNA和蛋白质等污染,完全适用于后继实验。同时对影响DDRT-PCR扩增的参数进行了筛选研究,确定了Mg2+、dNTP、引物和TaqDNA聚合酶的浓度和扩增程序。结果表明,在25μL DDRT-PCR反应总体系中各组分的最佳量分别为:17.2μL DEPC-ddH2O;1.5μL 10×PCR Buffer(Mg2+);2.0μL dNTP(各2.5mmol/L);1.0μL锚定引物H-T11M(10μmol);1.0μL随机引物(5μmol/L);1.5μL cDNA;0.8μLTaqDNA Polymerase(2.5 U/μL)。PCR反应程序:①94℃4 min;②30℃1 min 30 s;③72℃1 min;④94℃30 s;⑤42℃1 min 30 s;⑥72℃1 min;从④到⑥35个循环;⑦72℃10 min。

This study was conducted to develop a suitable method for extracting total RNA from stigma in self-compatible Yunjie. By using the improved guanidine thiocyanate method, total RNA from Yunjie stigmas were extracted and pure＆ The results showed that the total RNA were in good integrity and high purity, which demonstrated that RNA samples were free of contamination of protein, DNA, polysaccharide polyphenol, and were applicable to cDNA synthesis, DDRT-PCR. Furthermore, the factors affected DDRT-PCR were screened, a stable DDRT-PCR reaction system and process was set up,respectively. This system was 25 gL total volume contaited 17.2 uL DEPC-ddH2O; 1.5uL 10×PCR Buffer（Mg^2＋ ） ;2uL dNTP（each 2.5 mmol/L） ;1.0 uL H-T11M（10umol） ;1uL random primer（5 umol/L）;1. 5uLcDNA;0.8 uL Taq DNA polymerase（2.5U/ uL）. DDRT-PCR process as the follows;①94℃4 min;②30℃1 min 30 s;③72℃1 min;④94℃30 s;⑤42℃1 min 30 s;⑥72℃1 min;amplifying for 35 cycles from④to ⑥ ;（2）72℃10 min.