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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。
Using the Agrobacterium rhizogenes K599 (with the plasmid pBIN-35S-GFP), the in vitro cotyledon of cucumber inbred NC-46 with resistance genes against Meloidogyne incognita and Peanut root knot nematode as explants , Inducing transgenics adventitious roots efficiently (the induction rate was 76.7%). On this basis, a transgene adventitious root cDNA library was successfully constructed. The constructed cDNA library had a recombination rate of 98.3% and a primary library capacity of 2.02 × 106cfu. The gene integrity rate was 47.6% and that of Unigene was 66.7%. The selection of transgenic adventitious roots cDNA library, to overcome the seedling is difficult to obtain a large number of uniform root defects; to overcome the seedling roots by soil rhizosphere microorganisms, nematodes and other physiological conditions on the root of RNA contamination and extraction; use of Improved library construction of SmartcDNA library obtained clone contains full 5 ’untranslated region of the full-length cDNA; In addition, the construction of the library due to the use of improved pUC19 vector λTripIEx2 phage vector, the obtained clone no longer need to be inserted into The cDNA on the phage vector is transfected into the plasmid vector and can be used directly for hybridization and sequencing and therefore more convenient for gene cloning and screening.