摘要
将源自中国野生华东葡萄的抗病基因——病程相关蛋白基因导入‘砀山酥梨’,并对转化系统条件进行优化。首先将VpPR10基因重组于中间表达载体pWR-Ⅱ,得到重组质粒pWR-Ⅱ-VpPR10;继而以改进冻融法将其导入根癌农杆菌EHA105,再利用根癌农杆菌介导的叶盘转化法进行遗传转化。根癌农杆菌浓度OD600=0.5,离体叶片侵染5min,在含有100μmol·L-1乙酰丁香酮的培养基中暗处共培养48h有利于砀山酥梨的遗传转化;经PCR分析、Southern blot和RT-PCR检测,证明目的基因VpPR10已导入并整合到砀山酥梨基因组中;转化率为1.08%。
To provide the basis for increasing the genetic transformation efficiency of Pyrus bretshneideri‘Dangshan Suli＇,the VpPR10 gene,cloned from Chinese wild Vitis pseudoreticulata W.T.Wang,was introduced into pear genome and the optimum conditions of the genetic transformation system were also examined.The VpPR10 gene was constructed into the plant expression vector pWR-Ⅱ and the recombinant plasmid pWR-Ⅱ-VpPR10 was obtained;Then the pWR-Ⅱ-VpPR10 was introduced into the Agrobacterium tumefaciens strain EHA105 by improved freeze-thaw method,and was transformed into leaf disc explants of Dangshan Suli via an Agrobacterium tumefaciens-mediated system.The results showed that the suitable transformation conditions were 5 min infection time,0.5 bacterium concentration （OD600）,100 μmol · L-1 Acetosyringone（AS）,and 48 h co-cultivation in dark.The PCR,Southern blot and RT-PCR analysis results revealed that VpPR10 gene was successfully recombined into the genome of Dangshan Suli,and the rate of genetic transformation was 1.08%.
出处
《园艺学报》
CAS
CSCD
北大核心
2010年第10期1567-1574,共8页
Acta Horticulturae Sinica
基金
国家‘863’计划项目(2007AA10Z182)
陕西省重大科技专项(2006kz05-G4)
关键词
梨
抗病基因
农杆菌介导
病程相关蛋白基因(VpPR10)
条件优化
Pyrus bretschneideri Rehd.
disease-resistant gene
Agrobacterium tumefaciens-mediated
pathogenesis-related protein gene（VpPR10）
optimization
