甘蔗条纹花叶病毒HC-Pro、P3N-PIPO、CP和VPg基因酵母双杂交诱饵表达载体的构建及自激活检测

发布时间：2018-01-10 11:28

本文关键词：甘蔗条纹花叶病毒HC-Pro、P3N-PIPO、CP和VPg基因酵母双杂交诱饵表达载体的构建及自激活检测　出处：《华北农学报》2016年01期 　论文类型：期刊论文

【摘要】：为探索甘蔗条纹花叶病毒与甘蔗的互作机制,利用酵母双杂交技术筛选与SCSMV互作的甘蔗因子基因,利用PCR技术克隆了SCSMV的HC-Pro、P3N-PIPO、CP和VPg的编码区,构建到酵母双杂交诱饵载体p GBKT7上,获得了p GBKT7-HC-Pro、p GBKT7-P3N-PIPO、p GBKT7-CP和p GBKT7-VPg 4个诱饵载体。结果显示,将重组质粒转化酵母Y2HGold酵母菌株后,酵母菌株在SD/-Trp平板上生长良好,表明重组诱饵质粒表达产物对酵母细胞无毒性;在SD/-Trp/X-α-gal平板上长出白色菌落未变蓝,在SD/-Leu、SD/-Trp/-Ade、SD/-Trp/-His和SD/-Trp/X-α-gal/Ab A平板上不能生长,表明重组诱饵质粒表达产物对MEL1、ADE2、HIS3和AUR1-C报告基因无自激活作用。构建的诱饵表达载体可以作为诱饵用于文库筛选,为探索SCSMV侵染机制和发病机理奠定了基础。
[Abstract]:In order to explore the interaction mechanism between sugarcane stripe mosaic virus and sugarcane, the gene of sugarcane factor interacting with SCSMV was screened by yeast two-hybrid technique, and the HC-Pro of SCSMV was cloned by PCR technique. The coding region of CP and VPg was constructed into yeast two-hybrid bait vector p GBKT7 and p GBKT7-HC-Pro was obtained. Four decoy vectors, p GBKT7-P3N-PIPOP GBKT7-CP and p GBKT7-VPg, were obtained. After the recombinant plasmid was transformed into yeast Y2HGold yeast strain, the yeast strain grew well on the SD/-Trp plate, indicating that the recombinant bait plasmid expression product was not toxic to yeast cells. On the SD-Trp / X- 伪 -gal plate, the white colonies don't turn blue, and at the SD-Leuum SD-Trp / Ade. SD/-Trp/-His and SD-Trp / X- 伪 -gal-a-Ab A could not grow on the plate, indicating that the recombinant bait plasmid expressed product of MEL1 and ADE2. HIS3 and AUR1-C reporter genes have no self-activation. The constructed decoy expression vector can be used as bait for library screening, which lays a foundation for exploring the mechanism of SCSMV infection and pathogenesis.
【作者单位】：农业部福建甘蔗生物学与遗传育种重点实验室福建农林大学;
【基金】：国家高技术研究发展计划“863计划”项目(2013AA102604) 国家自然科学基金项目(31171605;31371688)
【分类号】：S435.661
【正文快照】： 甘蔗(Saccharum officinarum L.)是我国和全世界主要的糖料作物和能源作物。甘蔗花叶病(Sug-arcane mosaic disease,SMD)是严重危害甘蔗生产的病毒性病害之一,严重影响甘蔗的产量和品质。甘蔗感病后,病株叶片出现黄绿相间的斑纹、分蘖减少、生长缓慢、节间变短、品质下降,产量

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