炭疽菌侵染后刺葡萄果皮酵母双杂交cDNA文库构建

Yeast Two-hybrid cDNA Library Constructed from Vitis davidii Föex Pericarps Infected by Grape Ripe Rot Pathogen,Colletotrichum viniferum

【目的】炭疽病是南方葡萄的重要病害,刺葡萄是高抗炭疽病的重要野生葡萄资源,目前尚未见到关于葡萄炭疽菌侵染后的葡萄果皮酵母cDNA文库的报道。本文构建了葡萄炭疽菌侵染后刺葡萄果皮的酵母cDNA文库,为葡萄抗炭疽病分子机理研究提供材料基础。【方法】提取葡萄炭疽菌侵染后1、3、7 d的刺葡萄福安果皮总RNA,采用SMART的方法将总RNA反转录合成c DNA,使用试剂盒将cDNA纯化,随后将纯化产物(双链cDNA)酶切处理及短片段去除,获得质量良好的cDNA。将其克隆到pGADT7三框质粒载体,经纯化精制,获得初级c DNA文库。对初级cDNA文库扩增后,提取文库质粒转化到Y187酵母菌中,制作扩增的酵母文库,并对酵母文库进行鉴定。【结果】经检测所构建的初级cDNA文库库容约为5.2×10~6 cfu,重组率约为97.92%,插入的片段长度主要分布在400~2 000 bp,具有良好的多态性。进一步获得的Y187酵母文库滴度约为6.0×10~7 cfu·mL^(-1)。【结论】构建的葡萄炭疽菌诱导的刺葡萄果皮酵母cDNA文库质量符合酵母双杂交筛选要求,为筛选葡萄炭疽病侵染过程中的互作蛋白奠定基础。

【Objective】A yeast two-hybrid cDNA library on Colletotrichum viniferum-infected pericarps of the wild spine grape(Vitis davidii Föex),a cultivar highly resistant to the serious grape ripe rot in southern China,was constructed to facilitate studies on the molecular mechanism of the disease resistance in the plants.【Method】The total RNA was extracted from the pericarps of V.davidii accession,Fu'an,1,3,and 7d after C.viniferum inoculation and reverse-transcribed into cDNA using the SMART method.The cDNA was purified by an assay kit into a double stranded cDNA to be digested.Then,the short fragments were removed to obtain a high-quality cDNA for the subsequent cloning into a pGADT73-frame plasmid vector and purifying for the establishment of a primary cDNA library.After amplification,the plasmid was extracted and transformed into Yeast Y187 to produce the amplified library for final identification.【Result】The primary cDNA library had a capacity of approximately 5.2×106 cfu with a recombination rate of approximately 97.92%and the inserted fragments of good polymorphism in the lengths ranging from 400 to 2000 bp.The harvested Y187 yeast library titer was about 6.0×107 cfu·mL−1.【Conclusion】The constructed cDNA library from C.viniferum-infected grape pericarps was adequate for yeast two-hybrid screening.It would materially aid the studies on the interacting proteins in grape plants infected by the pathogen.