Construction of a full-length Enriched cDNA Library and Analysis of 3111 ESTs from Roots of Bupleurum chinense DC.

柴胡全長富集cDNA文庫構建及其3111個EST序列測定分析

隋春(Chun Sui) ； 魏建和(Jian-He Wei) ； 陳士林(Shi-Lin Chen) ； 陳懷瓊(Huai-Qiong Chen) ； 董樂萌(L. M. Dong) ； 楊成民(Cheng-Min Yang)

《Botanical Studies》 51卷1期 (2010/01) Pp. 7-16

摘要

中藥柴胡，爲柴胡屬植物的乾燥根，具有抗炎、退熱和保肝等功效。在中國、日本、韓國及南亞其他國家和地區廣泛應用。本文利用SMART技術構建了藥用柴胡屬植物北柴胡(Bupleurum chinense DC.)根的全長富集cDNA文庫，以啟動藥用柴胡功能基因組研究。文庫滴度爲1.1×10^6。從文庫中隨機選擇了3902個克隆進行5'端單反應測序，獲得了3111個高品質EST序列，包括377個序列重疊群(contigs)和1273個單一序列(singletons)共1650個獨立EST(uniESTs)。文庫插入片段平均長度約1.1kb，全長比率約51.5%。BlastX分析結果表明949(57.5%)個獨立EST和已鑒定基因同源，680(41.2%)個獨立EST與GenBank中未知、未命名或推測的蛋白基因同源，21個獨立EST沒有找到同源基因。獨立EST的GO(Gene Ontology)注釋顯示1002、957和861個獨立EST分別歸屬於細胞功能、生物過程和細胞組分三大類。與KEGG中擬南芥代謝途徑進行了比較分析，結果表明，307個獨立EST可能參與31個代謝途徑（每個代謝途徑至少包含5個獨立EST）。1650個獨立EST中82個含有總計86個微衛星位點(SSR)。本研究首次構建的柴胡根全長富集cDNA文庫及測序分析的EST克隆，爲研究這一中藥材的各種生理現象的分子基礎提供了有效平臺。從文庫資料中挖掘的具有潛在標記價值的SSR位點將爲柴胡種質鑒定，遺傳多樣性分析及基因定位提供有效分子標記。

關鍵字

北柴胡 ； 全長富集cDNA文庫 ； 表達序列標籤ESTs ； 技術 ； SSR

並列摘要

Radix Bupleuri (Chaihu), sourced from the dried roots of Bupleurum species, is a traditional Chinese medicine with anti-inflammatory, anti-pyretic, and anti-hepatotoxic efficacy. It is widely used in China, Japan, Korea, and other countries in south Asia. A full-length enriched cDNA library derived from the roots of B. chinense DC. was constructed for the first time by the SMART technique in this study to initiate the functional genomic research of this important medicinal plant. The titre of the library was 1.1×10^6. From the library, randomly selected 3902 clones were 5' single-pass sequenced, among which 3111 high quality ESTs were generated and 1650 uniESTs were identified with 377 contigs and 1273 singleton ESTs. The estimated average cDNA insert size was 1.1 kb, and the fullness ratio was ca. 51.5%. BlastX analysis of all uniESTs resulted in 949 (57.5%) homology to previously identified genes, 680 (41.2%) matched to unknown, unnamed, or hypothetical protein genes, and 21 clones had no hit. Gene ontology (GO) annotation of uniESTs showed that approximately 1002, 957, and 861 were assigned molecular function, biological process, and cellular component GO terms, respectively. KEGG pathway analysis indicated that 307 uniESTs may be involved in 31 metabolic pathways, in which at least five uniESTs were contained, by comparing each with Arabidopsis metabolic pathways. Through SSR searching within 1650 uniESTs, 86 potentially useful SSR loci were identified in 82 uniESTs. The library and EST data provide a platform to study the molecular mechanisms of various physiological phenomena of Bupleurum. The set of SSR loci would potentially be useful molecular markers for the germplasm identification, genetic diversity analysis, and gene mapping of Bupleurum.

並列關鍵字

Bupleurum chinense DC. ； Full-length enriched cDNA library ； ESTs ； Switching mechanism at 5' end of RNA transcript SMART ； SSR