丹参功能基因组学研究Ⅰ——cDNA芯片的构建  

Functional genomics studies of Salvia miltiorrhiza Establish cDNA microarray of S.miltiorrhiza

作　　者：崔光红[1] 黄璐琦[1] 唐晓晶[1] 邱德有[2] 王学勇[1] 付桂芳[1] 

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]中国林业科学院林业研究所,北京100091

出　　处：《中国中药杂志》2007年第12期1137-1141,共5页China Journal of Chinese Materia Medica

基　　金：国家重点基础研究发展计划(2006CB504700);国家高科技研究发展计划项目(2003AA2Z2040)

摘　　要：目的:构建丹参cDNA芯片,为基因表达谱研究提供条件。方法:采用CTAB法提取丹参根总RNA,采用Pharmacia公司QuichprepTMMicro mRNA Purifi Cation Kit分离mRNA后,通过在cDNA分子两端加上EcoRⅠ/NotⅠ接头,在T4多核苷酸激酶的作用下磷酸化,与表达载体λZAP Express Predigested Vector连接,然后用包装蛋白在体外包装连接产物,感染E.coliXL1-Blue MRF’构建成cDNA文库。挑取分离良好的噬菌斑进行PCR扩增,经过电泳检测、纯化、再次电泳检测后得到的克隆用于芯片点制。以丹参actin基因作为阳性对照,不含DNA的点样液和Poly A为阴性对照。结果:cDNA文库插入片段长度为500～2 500 bp。共得到4 354个克隆用于芯片点制。单色荧光(Cy3)杂交显示,阳性对照均出现杂交信号,阴性点没有杂交信号。整张芯片背景清晰,漏点少,杂交点为完整的圆形,芯片质量完好。结论:该芯片的制作为首张有关道地药材的cDNA芯片,为丹参功能基因组的研究提供了强有力的工具。Establishing cDNA microarray,in order to study functional genomics of Salvia miltiorrhiza. Method： Total RNA samples were prepared from S. miltiorrhiza roots using a modified CTAB method, mRNA was isolated by Quichprep^TM Micro mRNA Purification Kit from Pharmacia. Then cDNA was synthesized and cloned into the EcoRI-XhoI sites of the ZAP Express vector using a cDNA synthesis kit,and the ligation mixture was packaged using a ZAP-cDNA Gigapack Gold III cloning kit （Stratagene）. The single phage was isolated for PCR amplification,Aliquots of the PCR reactions were analyzed in a 1.5% agarose gel to verify the quality of PCR. The remaining cDNA was purified by Mtdtiscreen filter plates （MiUipore） and aliquots were analyzed by agarose gel again to verify the quality of purification. Clones passed verification was resuspended in 15 μL 50% DMSO for arraying. An actin gene from S. miltiorrhiza was used for positive control. PloyA and 50% DMSO was used for negative controls. Result： Bacterial colonies containing cNDAs of S. miltiorrhiza were inserted with average insert size of 0. 5 kb to 2. 5 kb. Total 4 354 genes were singled out from the first 8 736 PCR product and used for cDNA microarray manufacture. Single color fluorescence hybridization showed that all positive controls had signals while negative controls had no signals. Conclusion： It was the first cDNA microarray about traditional Chinese herbs esneciallv for geoherbs. It could be a oowerful tool for studying functional genomics of S. miltiorrhiza.

关 键 词：丹参 CDNA芯片 功能基因组学 

分 类 号：S567.53[农业科学—中草药栽培]