华北农学报2016，Vol.31Issue(4)：100-105,6.DOI:10.7668/hbnxb.2016.04.017
苹果抗 ASPV 病毒 RNAi 载体构建及转化砧木 M26的研究
Construction of Apple stem pitting virus Resistant RNAi Vector and Genetic Transformation into the Rootstock of M26
摘要
Abstract
In order to develop novel disease-resistant transgenic plant materials against ASPV,a virus resistant RNAi plant expression vector was constructed by the method of homologous recombination.With the Malus rootstock of M26,genetic transformation was conducted and positive transgenic plantlets were obtained successfully.Firstly,a conserved segment of coat protein gene of ASPV (489 bp)was cloned by PCR from cDNA of the virus-infective leaves of Royal gala.Then the fragment was inserted into the vector pENTR /SD /D by TOPO cloning technology to form the intermediate vector of pEN-ASPV.After this fragment was replaced and connected into the destination vec-tor pHellsgate12 by LR reaction,the RNAi plant expression vector Phe-ASPV was obtained.And the engineering bacteria EH-ASPV for plant transformation was obtained after the constructed RNAi vector was transformed into Agrobacterium strain EHA105 by alternate freezing and thawing method.By the Agrobaterium mediated method, transformation was conducted with the leaf discs of the Malus rootstock of M26.After culture and subculture for anti-biotics screening,bacteria elimination and rooting,compact kanamycin resistant plantlets formed from buds were ob-tained.After total RNA extracted from the well rooted plantlets,RT-PCR test was conducted and the result showed that the gene fragment of 489 bp carried on the RNAi structure had been transformed into the material of M26, which was expressed in plants on the level of RNA.Through this study,novel transgenic materials were obtained successfully,which laid foundation for disease-resistance evaluation by pathogen inoculation and virus-resistant mo-lecular breeding in the near future.关键词
苹果茎痘病毒/CP 基因/RNAi 载体/遗传转化Key words
Apple stem pitting virus/Coat protein gene/RNAi vector/Transgenosis分类
生物科学引用本文复制引用
田莉莉,牛良..苹果抗 ASPV 病毒 RNAi 载体构建及转化砧木 M26的研究[J].华北农学报,2016,31(4):100-105,6.基金项目
国家“863”项目 （）
