翻译和非翻译马铃薯Y病毒外壳蛋白基因介导的抗病性比较  

Expression of Translatable and Untranslatable Potato Virus Y(PVY^N) Coat Protein(CP)Genes in Tobacco and Comparison of Virus Resistance Between the Transgenic Plants

作　　者：郭兴启[1] 温孚江[1] 宋云枝[1] 孟祥兵[1] 吕士恩[1] 郑成超[1] 朱常香[1] 

机构地区：[1]山东农业大学生命科学学院,山东泰安271018

出　　处：《病毒学报》2001年第4期360-367,共8页Chinese Journal of Virology

基　　金：国家自然科学基金 ( 39970 485 ) ;山东省自然科学基金( 2 2 0 0 0D0 2 )

摘　　要：利用RT PCR方法克隆获得马铃薯Y病毒烟草叶脉坏死株系 (PVYN)的可翻译和不可翻译外壳蛋白 (CP)基因 ,并分别插入 pROKⅡ质粒中获得重组双元表达载体。通过根癌农杆菌(Agrobacteriumtumefaciens) 介导的基因转化方法 ,将可翻译和不可翻译的PVYN CP基因分别导入烟草栽培品种NC89叶片组织中 ,得到抗卡那霉素的再生植株。对所得到的抗卡那霉素的再生苗进行PCR检测表明 ,被导入可翻译和不可翻译CP基因的植株分别占卡那霉素抗性植株的 95 %和 98%。攻毒实验表明 ,两种类型的转基因烟草对PVYN 的抗病性具有相似性 ,其表现型为 :免疫、抗病和感病。免疫型转基因植株的抗病性不受接种物类型及其剂量的影响。Southern印迹杂交结果显示 ,目的基因已经整合到烟草基因组中。Northern印迹杂交证明 ,两种类型的CP基因都已在RNA水平上得到了表达 ,但细胞内RNA的积累量与转基因植株的抗病强度成负相关。Western印迹杂交表明 ,在表达不可翻译PVYN CP基因的转基因植株内未检测到CP蛋白 ,而在导入可翻译的PVYN CP基因的植株内检测到了CP蛋白 ,且CP蛋白含量与抗病性不存在正相关。本研究结果证明 ,表达可翻译与不可翻译PVYN CP基因的转基因烟草对PVYN 的抗病性均为RNA介导的抗病性。The translatable and untranslatable coat protein(CP)genes of potato virus Y(tobacco veinal necrosis strain,PVY N)were introduced into tobacco(NC89)plants via Agrobacterium tumefaciens mediated transformation.Transformed tissues were selected in the presence of 100μg/ml Kanamycin sulfate and regeneration plants initially were screened by PCR.The results indicated that 95% of the plants transformed with translatable PVY N CP gene and 98% of the plants transformed with untranslatable PVY N CP gene were PCR positive.Southern blot analysis confirmed that the foreign gene had been integrated into the tobacco chromosomes.The transgenic plants were tested for resistance against PVY N infection in greenhouse.We observed three different responses to virus infection:1)some were immune,and no virus replication was detected;2)some were resistant(light symptoms were shown up 20-30 days post inoculation);and 3)some were susceptible to PVY N infection.Moreover,the resistance of immune plants was not overcomed by viral inoculation,either with PVY N-RNA inoculation or with PVY N particles as inoculum.Gene expression was verified by Northern or Western blot.An inverse correlation between transgene transcript accumulation and virus resistance was often noted when translatable or untranslatable PVY N CP genes were expressed.As expected,no PVY N CP was detected in the plants transformed with untranslatable PVY N CP gene,and the plants transformed with translatable PVY N CP gene contained PVY N CP,but there was no correlation between the accumulation of the CP and the resistance level.These results suggest that the resistance observed in the two types of the transgenic plants were based on the presence of PVY N CP RNA sequences rather than the accumulation of viral coat protein.

关 键 词：马铃薯Y病毒 外壳蛋白基因 转基因烟草 RNA介导 抗病性 CP 翻译 

分 类 号：S572[农业科学—烟草工业] S435.72[农业科学—作物学]