黄瓜多抗自交系NC-46转基因不定根的高频诱导及其cDNA文库的构建  

Induction of Hairy Root on Cucumber Inbred Line NC-46 with High Frequency and Construction of a Hairy Root cDNA Library

作　　者：孟莎莎[1] 向太和[1] 王琳[1] 

机构地区：[1]杭州师范大学生命与环境科学学院,杭州310036

出　　处：《园艺学报》2010年第4期567-574,共8页Acta Horticulturae Sinica

基　　金：浙江省自然科学基金项目(Y3080184);杭州市科技发展计划项目(20091133B07)

摘　　要：利用发根农杆菌K599(带有质粒pBIN-35S-GFP),以具有抗爪哇根结线虫和花生根结线虫等多种抗性基因的黄瓜自交系NC-46离体子叶为外植体,高效诱导出转基因不定根(诱导率达76.7%);并在此基础上成功构建了转基因不定根的cDNA文库。构建的cDNA文库重组率为98.3%,原始库容量达2.02×106cfu,其中基因的完整性比率为47.6%,Unigene比例为66.7%。选用转基因不定根构建根的cDNA文库,克服了实生苗难以获得大量均一状态根的缺陷;克服了实生苗根受到土壤根际微生物、线虫等对根生理状态的影响以及对提取RNA的污染问题;利用改良的SmartcDNA文库构建法,获得的文库克隆含有完整的5′端非翻译区的全长cDNA;此外,构建的文库由于利用改良的pUC19载体替代了λTripIEx2噬菌体载体,获得的克隆无需再将插入到噬菌体载体上的cDNA转染到质粒载体,可直接用于杂交和测序筛选,因此在基因克隆筛选时更加方便。Cucumber（Cucumis sativus）inbred line NC-46 contains genes that confer resistance to pathogens including Meloidogyne javanica and Meloidogyne arenaria. Hairy root of NC-46 was induced from cotyledon by wild-type Agrobacterium rhizogenes K599 harboring pBIN-35S-GFP with a frequency of 76.7%. A cDNA library of the hairy root of the transgenic cucumber was constructed successfully. The percentage of recombinant clones in the cDNA library was 98.3%. The capacity of the cDNA library reached 2.02 × 106 cfu,and the percentages of full-length gene and Unigene were 47.6% and 66.7%, respectively. Used of the hairy root of the transgenic cucumber for cDNA library construction overcame the difficulty to obtain a large amount of homogeneous natural roots,the varied physiological states of the roots caused by soil microbes,nematode and RNA contamination. With the modified Smart cDNA construction method,the cloned genes in the cDNA library contain the full length of 5′-introns. Moreover,the cDNAs were inserted into a developed pUC19 plasmid instead of λTripIEx2 phage vector. The cDNA containing plasmid can be used directly for hybridization and sequencing,facilitating gene screening. The cDNA library is useful for the screening of pathogen resistant genes and the cloning of genes involved in hairy root development.

关 键 词：黄瓜 转基因 毛状根 CDNA文库 构建 

分 类 号：S642.2[农业科学—蔬菜学]