感染高粱花叶病毒甘蔗叶片cDNA文库构建及评价

为了研究甘蔗花叶病与甘蔗寄主致病的互作分子机制,利用SMART技术成功构建了感染高梁花叶病毒甘蔗叶片的cDNA文库,用于后续酵母双杂交互作蛋白筛选试验.采用Omega公司Plant RNA Kit提取感染高粱花叶病毒甘蔗叶片总RNA,经过Oligotex纯化获得mRNA,将其反转录成cDNA第一链,再在DNA聚合酶作用下,通过长距离PCR扩增双链cDNA.经SfiI酶切并去除短片段后,连接到pGADT7-SfiI载体上,成功获得初级cDNA文库,最后以初级文库100万克隆为基数扩增,得到扩增文库并提取质粒.经检测构建的文库容量为1.6×106 cfu,文库滴度2.2× 106 cfu/mL,文库cDNA插入片段长度主要分布在700～2 000 bp,文库重组率约为96％.结果表明,该文库质量较好,为筛选分离抗病功能基因及开展寄主与病毒互作的研究奠定了基础.

Construction and Evaluation of Yeast Two Hybrid cDNA Library of Suagarcane Leaf Infected SrMV Virus

Sugarcane(Saccharum officinarum L.)is one of important specie producing sugar and energy cash crop.It plays an important role in sugar jar of raw materials and increases farmers’ income.Total RNA was extracted from infected SrMV sugarcane leaf using Omega company RNA extraction reagent.Total RNA was used to purify mRNA with Oligotex kit.The first strand cDNA was synthesized by reverse transcription of mRNA with SMART technique and LD-PCR was performed to synthesize double strand cDNA.Then double strand cDNA were digested by SfiI enzyme to remove shorter cDNA by running through a CHROMA SPIN TE-400 column.Purified ds cDNA and linear vector pGADT7-SfiI co-transformed into E.coli XL10-GOLD strain to generate sugarcane leaf infected SrMV virus’s Yeast-Two-Hybrid primary cDNA library.Then we obtained the amplified library and extracted the plasmids.The results of detection showed that the library contained 1.6×106 independent clones,and the titer of library were 2.2×106 cfu/mL.The sizes of most inserts were from 700 to 2 000 bp in the library.The recombination rate of library was 96%.These results indicated that the library was suitable for screening interaction proteins with SrMV coded proteins,further to understand the pathogenic molecular mechanisms of host resistance and exploit resistant germplasm resources.