摘要
水稻条纹叶枯病是以灰飞虱(Laodelphax striatellus)为传播介体的间歇性水稻病毒流行病害,流行年份造成水稻生产严重损失。在对该病病原水稻条纹叶枯病毒(RSV)外壳蛋白进行了氨基酸组成、cDNA合成、克隆、产物表达和全序列分析基础上,将RSV外壳蛋白基因插入植物表达载体pROK Ⅱ,用基因枪法转化水稻悬浮细胞系。轰击前将分散性好的胚性细胞团均匀地铺在含有滤纸的平皿上。用CaCl_2和亚精胺将质粒DNA包裹于平均直径为1.09μm的钨粉上。将钨粉滴在子弹前部凹穴中,在-0.095 MPa真空度的条件下击发。在存在G418条件下,两个月后可从褐化培养物中筛选出一些抗性细胞团。将其转入分化培养基后分化出绿苗。用Southern杂交法测定了由抗性细胞团再生的10株水稻苗,其中有2株出现了分子量与CP基因对应的杂交带。Western测定的结果显示,在转化植株中有CP基因的表达。
Rice stripe virus (RSV) is a pathogen of rice stripe disease causing great damage to rice.The disease is transmitted by Laodelphax striatellus and three other planthoppers.RSV infects as much as 37 cereals including rice,wheat,maize and results in a significant reduction in yield in epidemic year.In order to develop efficient means of controlling the disease, authors have studied the amino acid composition of RSV coat protein (CP),synthesized and cloned the cDNA to CP,sequenced the full-length CP gene.Having inserted the RSV CP gene into plant expression vector pROK II,authors transformed rice suspension culture via microprojeqtile bombardment and obtained transgenic plants expressing the CP gene.The suspension culture was initiated by inoculating yellowish,compact and embryogenic calli derived from seeds into suspension medium containing proline and maltose.After being cultured at 26℃ in the dark for about half a year,finely-dispersed and embryogenic suspension culture was estabolished.Before bombardment the suspension culture was evently applied onto three-layered filter-paper discs in a petri dish.CaCl_2 and spermidine was employed to coat tungsten particle with plasmid DNA.2.5 μl of coated particle was loaded onto bullet and each dish was bombarded three times.Immediately after being bombarded,the suspensions were cultured in modified Ns medium.2 days later the suspensions were transferred to the same medium but containing G418,which were subcultured weekly.Being subject to G418 selection for two months,white and fast-growing clones were emerged from the brownish cultures. Green plants regenerated when the resistant calli were transferred to differentiation medium.The regenerated plants were firm enough to grow well in the greenhouse.10 plants regenerated from G418 resistant calli were tested for their transformed nature by Southern blot using ^(32)P-labelled CP gene as a probe.Among the plants tested,2 plants showed clearly hybridizing bands with a molecular weight corresponding to RSV CP gene.Western blot further demonstrated that RSV CP gene was expressed in transgenic rice plants.At present tests on the antiviral effects of transgenic plants by feeding plantphoppers infected with RSV are being underway.
基金
洛氏基金水稻生物技术项目资助课题(RF 90032,#69)
关键词
水稻
病毒
外壳蛋白
基因表达
Oryza sativa
Transformation
Virus
Coat Protein
