用λ－ZAPⅡ作载体构建豌豆卷须cDNA文库

Construetion of cDNA Library from Pea Tendrils with the Veetor Lambda ZAP Ⅱ

用改进的异硫氰酸胍－苯酚－氯仿抽提法提取了豌豆卷须总ＲＮＡ并纯化了ｍＲＮＡ，合成双链ｃＤＮＡ后加上人工接头，Ｓｅｐｈａｒｏｓｅ ２Ｂ柱层板去掉小片段，最后连入载体λ－ＺＡＰⅡ并用噬菌体包装蛋白进行体外包装，经稀释测定出含有重组子的文库大小为９．２×１０＾５，原位杂交筛选出了肌动蛋白阳性克隆。

Total RNA was isolated from two-week-old pea (Pisum sativum) tendrils by the method of guanidine isocyanate-phenol-chloroform extraction. Poly￣ (A+ ) RNAs were selected by affinity chromatography through an oligo (dT)cellulose column. The cDNAs were synthesized by reverse transcription with Oligo(dT)as the primer and α-32P-dCTP as an indicatior of DNA synthesis. Autoradiograph of ￣(32)P-labelled cDNA on NaOH gel indicated that the length of cDNA was up to 8000 bp. The cDNA was ligated to Not Ⅰ/EcoR Ⅰ adaptor and fractionated through a sepharose ZB column. The collected fragments ranged from 300 bp to 8000 bp were inserted into Lambda-ZAP Ⅱ/EcoR Ⅰ site by ligation, and then packaged in vitro into viron particles with packaging protein extracts. This cDNA library contained 9. 2X105 recombinant phage,as determined on X-gal/IPTG plates. By in situ plaque hybridization ,eighteen positive clones were identified using yeast actin gene as a probe. These positive clones were used for in vivo excision and recombinant plasmids containing actin cDNAs were obtained. The sequencing of these pea tendril actin cDNA clones is under way.