苏俄苹果酵母双杂交cDNA文库的构建与评价  

Construction and evaluation of yeast two-hybrid cDNA library of Malus sylvestris cv.R12740-7A

作　　者：段豪[1] 赵绪生[1] 钟敏[1] 胡同乐[1] 王树桐[1] 张瑜[1] 张珣[2] 王亚南[1] 曹克强[1] 

机构地区：[1]河北农业大学植物保护学院,河北保定071001 [2]沈阳大学环境学院,辽宁沈阳110044

出　　处：《河北农业大学学报》2014年第1期69-73,共5页Journal of Hebei Agricultural University

基　　金：国家自然科学基金资助项目(31201487;41001185);河北省自然科学基金资助项目(C2013204058)

摘　　要：以苏俄苹果(Malus sylvestris cv.R12740-7A)组培苗为试材,构建了以真核表达载体pGADT7为基础的苹果酵母双杂交cDNA文库。通过异硫氰酸胍法提取总RNA,Oligotex-dT30<Super>mRNA Purification Kit分离纯化mRNA,利用SMART法反转录合成双链cDNA(dscDNA),酶切纯化后的dscDNA与库载体pGADT7连接电转大肠杆菌,经涂板检测,初始文库滴度为2.67×105 cfu/mL,库容量为4.0×106 cfu,随后将文库进行了百万扩增,扩增文库滴度为1.0×108 cfu/mL,库容为1.5×1010 cfu,在文库中随机挑取46个克隆,利用PCR方法测得文库插入片段75%大于1kb,重组率为100%,文库质量较高,符合文库构建的标准,为后续苹果分子生物学研究以及病毒与寄主互作研究奠定了基础。The eDNA Library of apple was established on the basis of the eukaryotie expression vectors pGADTT, using Russia apple （Malus sylvestris cv. R12740-7A） seedlings as material. Total RNA was extracted by the guanidinium thiocyanate method. Oligotex-dT30 〈Super〉 mRNA Purification Kit was used to separate and purify the mRNA. Double-stranded eDNA （dscDNA） was produced using the SMART method of reverse transcription. After enzyme digestion and purification, dscDNA was connected with vector pGADT7 and electrically trans- formed to Escherichia coll. The titers and the capacity of the primary library were 2.67×10^5 cfu/mL and 4.0×10^8 clones. After millions of amplification, the amplified library titer was 1.0×10^8 cfu/mL and the capacity was 1.5× 10^l0 clones. Forty-six clones were randomly picked from the library and 75% of eDNA insert fragments were measured more than lkb by using the PCR method. The recombination rate was 100%. The library is of high quality and meets with the standard of cDNA library, which established a basis for the research of apple molecular bi- ology and the interaction between virus and host in the following study.

关 键 词：苏俄苹果 酵母双杂交cDNA文库 构建 

分 类 号：S432.41[农业科学—植物病理学]