Namba S, Ling K S, Gonsalves C, Gonsalves D, Slightom J L
Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva 14456.
Gene. 1991 Nov 15;107(2):181-8. doi: 10.1016/0378-1119(91)90317-5.
The gene (cp) encoding the coat protein (CP) of cucumber mosaic virus (CMV) strain WL (CMV-WL, which belongs to CMV subgroup II) was custom polymerase chain reaction (CPCR)-engineered for expression as described by Slightom [Gene 100 (1991) 251-255]. CPCR amplification was used to add 5'- and 3'-flanking NcoI sites to the CMV-WL cp gene, and cp was cloned into the expression vector, pUC18cpexp. This CMV-WL cp expression cassette was transferred into the genome of tobacco (Nicotiana tabacum cv. Havana 423) via the Agrobacterium T-DNA transfer mechanism. R0 plants that express the CMV-WL cp gene were subcloned, propagated, and challenge-inoculated with CMV-WL. Several R0 plant lines showed excellent protection against CMV-WL infection; however, plants found to accumulate the highest CP levels did not show the highest degree of protection. Thus in our case, CP levels appear not to be a useful predictor of the degree of protection. Plants from the best protected CMV-WL cp gene-expressing R0 tobacco lines were also inoculated with CMV strains belonging to the other major CMV subgroup (subgroup I), CMV-C and CMV-Chi, and compared in a parallel experiment with a transgenic tobacco plant line that expresses the CMV-C cp gene. Plants expressing the CMV-WL cp gene appeared to show a broader spectrum of protection against infection by the various CMV strains than plants expressing the CMV-C cp gene.
黄瓜花叶病毒(CMV)WL株系(CMV-WL,属于CMV亚组II)的外壳蛋白(CP)编码基因(cp)按照Slightom [Gene 100 (1991) 251-255]所述,通过定制聚合酶链反应(CPCR)进行工程改造以用于表达。利用CPCR扩增在CMV-WL cp基因的5'和3'侧翼添加NcoI位点,并将cp克隆到表达载体pUC18cpexp中。通过农杆菌T-DNA转移机制将这个CMV-WL cp表达盒转入烟草(Nicotiana tabacum cv. Havana 423)基因组中。对表达CMV-WL cp基因的R0植株进行亚克隆、繁殖,并接种CMV-WL进行挑战接种。几个R0株系对CMV-WL感染表现出优异的抗性;然而,发现积累最高CP水平的植株并未表现出最高程度的抗性。因此,在我们的研究中,CP水平似乎不是抗性程度的有效预测指标。来自最佳抗性的表达CMV-WL cp基因的R0烟草株系的植株也接种了属于其他主要CMV亚组(亚组I)的CMV株系,即CMV-C和CMV-Chi,并在平行实验中与表达CMV-C cp基因的转基因烟草株系进行比较。与表达CMV-C cp基因的植株相比,表达CMV-WL cp基因的植株似乎对各种CMV株系的感染表现出更广泛的抗性谱。