| 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 |
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| 引用本文： | 白生文,孙万仓,范惠玲,张芬琴,肖占文,程红玉,孙晓岩.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[J].西北农业学报,2009,18(4):140-143. |
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| 作者姓名： | 白生文 孙万仓 范惠玲 张芬琴 肖占文 程红玉 孙晓岩 |
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| 作者单位： | 白生文,张芬琴(河西学院,生命科学与工程系,张掖,734000);孙万仓(甘肃农业大学农学院,兰州,730070);范惠玲,肖占文,程红玉,孙晓岩(河西学院,农学系,张掖,734000) |
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| 基金项目： | 芸芥自交亲和基因的克隆及功能分析项目 |
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| 摘 要： | 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Taq DNA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;1.5μL10×PCR Buffer(Mg2+);2.0μL dNTP(各2.5mmol/L);1.0μL锚定引物H-T11 M(10 μmol);1.0μL随机引物(5μmol/L);1.5μL cDNA;0.8 μL TaqDNA 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℃1min 30 s;⑥72℃1 min;从④到⑥35个循环;⑦72℃10 min.
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| 关 键 词： | 芸芥 提取方法 反应体系 |
| 收稿时间： | 2008/12/24 0:00:00 |
| 修稿时间： | 2009/5/12 0:00:00 |
Studies on Methods for Extracting Total RNA from Stigmas and mRNA Reaction System for Screening Genes Related to Self-compatibility in E.Sativa Mill. |
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| BAI Shengwen,SUN Wancang,FAN Huiling,ZHANG Fenqin,XIAO Zhanwen,CHENG Hongyu and SUN Xiaoyan.Studies on Methods for Extracting Total RNA from Stigmas and mRNA Reaction System for Screening Genes Related to Self-compatibility in E.Sativa Mill.[J].Acta Agriculturae Boreali-occidentalis Sinica,2009,18(4):140-143. |
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| Authors: | BAI Shengwen SUN Wancang FAN Huiling ZHANG Fenqin XIAO Zhanwen CHENG Hongyu and SUN Xiaoyan |
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| Institution: | Department of Bioscience and Biotechnology, Hexi University, Zhangye Gansu 734000, China;College of Agronomy, Gansu Agricultural University;Lanzhou 730070, China;Department of Agronomy, Hexi University, Zhangye Gansu 734000, China;Department of Bioscience and Biotechnology, Hexi University, Zhangye Gansu 734000, China;Department of Agronomy, Hexi University, Zhangye Gansu 734000, China;Department of Agronomy, Hexi University, Zhangye Gansu 734000, China;Department of Agronomy, Hexi University, Zhangye Gansu 734000, China |
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| Abstract: | 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 pured.The results showed that the total RNA were in good integrity and high purity, which demonst rated 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. |
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| Keywords: | DDRT-PCR |
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