Cui Guang-hong, Huang Lu-qi, Qiu De-you, Yuan Yuan, Fu Gui-fang
Institute of Chinese Materia Medica, Academy of Chinese Medical Sciences, Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2007 Jul;32(13):1267-72.
Studying the gene expression profiling of different stage hairy root of Salvia miltiorrhiza, in order to find functional genes.
The contents of second metabolites were determined by HPLC and gene expression profiling was detected by cDNA microarray. cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine's linear RNA amplification method and subsequent enzymatic reaction. The microarrays were scanned with a ScanArray Express scanner using ScanArray 2.0 software and quantified by signal intensities of individual spots from the 16-bit TIFF images using GenePix Pro 4.0. The linear normalization method was used for data analyze. Northern blot was used to test the gene expression results obtained by microarray. Different expressed genes were sequenced and analyzed by gap4 software, and then they were analyzed with BLASTX, BLASTN, GO and KEGG.
Growth rate and second metabolites analysis indicated that the stage from 30 d to 45 d was the growth stage, while the stage from 45 d to 60 d was the second metabolites accumulation stage. Accordingly 30 d hairy root was chosen as a reference, which was hybridized with 45 d and 60 d hairy root separately. Total 203 different expressed genes were obtained. Northern blot showed that the result was identical with the microarray result. After sequenced, there were 172 genes clustered into 114 clusters (Unigenes). Among them, 62 unigenes had known functions, 34 unigenes were hypothetical protein, 9 unigenes were homologues with no similarity and 9 unigenes were unidentified protein with low similarity. Total 67 genes were classified into cellular component ontology, molecular function ontology and biological process ontology based on GO analysis. Total 26 genes, which represented 29 metabolic-related enzymes, were located in metabolic maps based on KEGG pathway classification.
Several important functional genes related to second metabolite synthesis were cloned such as P450 and copalyl diphosphate synthase genes. cDNA microarray was a useful tool for functional genomics of traditional Chinese medicine.
研究丹参不同生长阶段毛状根的基因表达谱，以寻找功能基因。
采用高效液相色谱法测定次生代谢产物含量，利用cDNA微阵列检测基因表达谱。通过Eberwine线性RNA扩增法及后续酶促反应制备用荧光染料（Cy5和Cy3-dCTP）标记的cDNA。使用ScanArray 2.0软件在ScanArray Express扫描仪上扫描微阵列，并使用GenePix Pro 4.0通过16位TIFF图像中各个点的信号强度进行定量。采用线性归一化方法进行数据分析。用Northern印迹法检测微阵列获得的基因表达结果。对差异表达基因进行测序并用gap4软件分析，然后用BLASTX、BLASTN、GO和KEGG进行分析。
生长速率和次生代谢产物分析表明，30天至45天为生长阶段，45天至60天为次生代谢产物积累阶段。因此选择30天的毛状根作为对照，分别与45天和60天的毛状根进行杂交。共获得203个差异表达基因。Northern印迹显示结果与微阵列结果一致。测序后，172个基因聚集成114个簇（单基因簇）。其中，62个单基因簇具有已知功能，34个单基因簇为假定蛋白，9个单基因簇为无相似性的同源物，9个单基因簇为低相似性的未鉴定蛋白。基于GO分析，共67个基因被分类到细胞组分本体、分子功能本体和生物学过程本体中。基于KEGG途径分类，共有26个基因（代表29种代谢相关酶）定位在代谢图谱中。
克隆了几个与次生代谢产物合成相关的重要功能基因，如细胞色素P450和柯巴基二磷酸合酶基因。cDNA微阵列是中药功能基因组学的一种有用工具。
