丹参功能基因组学研究Ⅱ——丹参毛状根不同时期基因表达谱分析

目的：研究丹参毛状根不同时期的基因表达谱,发掘丹参功能基因。方法：通过比较不同时期丹参毛状根的生长量和次生代谢产物含量确定用于cDNA芯片杂交的材料。采用间接标记法进行探针标记后进行杂交反应,GenePix Pro 4.0软件对芯片图像进行分析,数据用Lowess方法进行归一化处理。采用两倍差异标准结合T检验来确定差异表达基因。Northern点杂交检验4个基因与芯片杂交结果的一致性。差异基因单向测序后经过gap4软件拼接聚类,然后利用BLASTX,BLASTN进行比对分析,利用Gene Ontology和KEGG进一步预测基因的功能。结果：30~45 d为丹参毛状根的快速增殖期,45~60 d为丹参次生代谢产物的快速积累期。将45,60 d丹参毛状根分别与30 d材料进行杂交,得到203个差异基因。4个基因Northern点杂交的结果与芯片杂交结果一致。测序后得到172条EST,拼接聚类后形成114个Unigene,其中功能已知基因62个,功能未定的假想蛋白34个,相似性较低的未鉴定基因9个,无显著相似性的基因9个。 “GO”分类中67个基因得到注释,KEGG分析中74个基因得到注释,26个有详细的代谢途径分析。结论：得到一系列次生代谢相关基因如细胞色素P450、二萜合酶等基因片段,为深入开展丹参功能基因组学研究提供了基础。

Functional genomics studies of Salvia miltiorrhiza Ⅱ——gene expression profiling of different stage of hairy root

Objective: Studying the gene expression profiling of different stage hairy root of Salvia miltiorrhiza,in order to find functional genes.Method: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.Result: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.Conclusion: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.