Characterization of Transgenic Tobacco Plants Expressing the Coat Protein Gene of Cucumber Mosaic Virus from Taiwan

本省分離之胡瓜嵌紋病毒鞘蛋白基因轉型菸草特性之研究

陳富永(Fuh-Yung Chen) ； 葉錫東(Shyi-Dong Yeh)

《植物病理學會刊》 3卷4期 (1994/12) Pp. 186-197

https://doi.org/10.6649/PPB.199412_3(4).0002

摘要

胡瓜嵌紋病毒(cucumber mosaic virus，CMV)為本省茄科﹑豆科、葫蘆科等作物栽培上的重耍問題，成為菸草、番茄、甜椒產量上的主要限制因子之一，在無抗病材料可利用下，問題難解決。為解決此難纏的病毒病害，本實驗即藉由遺傳工程方式，嘗試構築胡瓜嵌紋病毒鞘蛋白(coat protein，CP)的轉型菸草，以期對此病毒產生抗性。自本省分離之胡瓜嵌紋病毒番茄分離株CMV-T之鞘蛋白基因先經聚合鏈鎖反應(PCR)放大後加以選殖﹐再轉移至二位元農桿菌載體Agrobacterium經其媒介轉殖於菸草中。在所得之二十八個磚型株系(transgenic Iines)中，轉型植株復經聚合鏈鎖反應(PCR)確定其染色體中確含有CMV之CP基因。復以西方轉漬法(Western blotting)及間接聯血清吸附法(indirect ELISA)偵測得三株系表現較強，其它則表現較弱不易測得。所得轉型植株中分別以四個株系及五個株系在冬夏兩季進行兩次接種CMV-T試驗，結果顯示這些轉型植株均可明顯地延遲病徵表現，甚至在整個冬季試驗過程中，有些轉型植株並不產生病徵。綜合上述CMV CP轉型菸草構築成功之結果，可預期此遺傳工程保護方式將可應用到其發作物上，構築鞘蛋白轉型植株，以期對CMV的感染產生抗性。

關鍵字

胡瓜嵌紋病毒 ； 鞘蛋白 ； 轉型植物 ； 遺傳工程

並列摘要

Cucumber mosaic virus (CMV) causes great losses in several crops in Taiwan, especially in tobacco, tomato and pepper, Lack of resistant source makes the problem difficult to solve. Thus, CP-mediated protection provides an innovative strategy to control CMV in the crops. The objective of this study was directed to construct and evaluate transgenic tobacco plants expressing the CP of a local tomato-infecting CMV-T isolate as the first step for testing the feasibility of the approach. The coat protein (CP) gene of the virus was cloned from the DNA fragment amplified by polymerase chain reaction (PCR) and further constructed in a Ti-binary vector. Twenty eight putative transgenic plant lines of Nicotiana tabacum cv. Havana that expressing the CP of CNIV-T were produced via Agrobacterium-mediated transformation. The integration and expression of the CP gene was confirmed by PCR, Western blotting and indirect ELISA. Three transgenic lines expressed the CP gene at higher levels as reflected in ELISA and Western blotting. Resistance of the transgenic lines against CMV-T infection was evaluated by mechanical inoculation with the virus.. Four and five transgenic lines were subjected to challenge inoculations in winter and summer, respectively. In general, the transgenic lines showed a significant delay in symptom development, and the higher expressors showed better resistance than the lower expressors, Some plants of lines 1-10, 1-23, and 1-28, and all plants of line 1-17 did not show any symptoms when tested under low inoculum pressure and low temperature conditions. The production of CP transgenic tobacco resistant to CMV infection provides a model system for control of the diseases caused by CMV in other crops.

並列關鍵字

cucumber mosaic virus ； transgenic plant ； CP-mediated protection