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

园艺学报 ›› 2010, Vol. 37 ›› Issue (4): 567-574.

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

孟莎莎，向太和*，王琳

（杭州师范大学生命与环境科学学院，杭州310036）

收稿日期:2010-01-08 修回日期:2010-04-02 出版日期:2010-04-25 发布日期:2010-04-25
通讯作者: 向太和

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

MENG Sha-sha，XIANG Tai-he*，and WANG Lin

（College of Life and Environment Sciences，Hangzhou Normal University，Hangzhou 310036，China）

Received:2010-01-08 Revised:2010-04-02 Online:2010-04-25 Published:2010-04-25
Contact: XIANG Tai-he

摘要/Abstract

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 × 106 cfu，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Smart cDNA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。

关键词: 黄瓜, 转基因, 毛状根, cDNA文库, 构建

Abstract:

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.

Key words: cucumber, transgenic, hairy root, cDNA library, construction

中图分类号:

S 642.2

孟莎莎;向太和;王琳. 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[J]. 园艺学报, 2010, 37(4): 567-574.

MENG Sha-sha;XIANG Tai-he;and WANG Lin. Induction of Hairy Root on Cucumber Inbred Line NC-46 with High Frequency and Construction of a Hairy Root cDNA Library[J]. ACTA HORTICULTURAE SINICA, 2010, 37(4): 567-574.

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