Coat Protein-Mediated Resistance to Tomato Mosaic Virus in Systemic and Local Lesion Hosts

局部病斑寄主及系統性寄主鞘蛋白轉基因煙草植物對番茄嵌紋病毒之抗性

葉錫東(Shyi-Dong Yeh) ； 彭瑞菊(Jui-Chu Pon)

《植物病理學會刊》 5卷1期 (1996/03) Pp. 15-27

https://doi.org/10.6649/PPB.199603_5(1).0002

摘要

番茄嵌紋病毒(ToMV)在台灣的番茄及甜椒栽培區感染相當嚴重，此一病毒病害為值物病害中極需解決的問題，本實驗之目的即在選殖ToMV之鞘蛋白基因，解出其核苷酸序列，以了解其分子特性；並用以構築ToMV鞘蛋白轉基因植物，以進行遺傳工程保護試驗。由本省番茄田中分離得到的ToMV，大量純化後，再分離出病毒之RNA，藉由二個對應ToMV CP之引子，利用聚合酶鏈反應(PCR)之技術，得到對應ToMV RNA模板3'端的選殖株pTCP，此選殖株之限制酶片段再加選殖， 以雙去氧核苷酸鏈終止法解析，解出其核酸序列，結果顯示cDNA全長690個核苷酸包含全部鞘蛋白基因及3'端非轉譯區，而且只有一個轉譯架倫(open reading frame)，可對映產生一含158 個胺基酸，分子量為17.5 kDa的蛋白，與日本之ToMV相較，在CP上有3個核苷酸不同，但胺基酸序列則完全個同，顯示台灣之ToMV和日本之ToMV 幾近相同。pTCP在生體外(in vitro) 進行轉錄及轉譯作用，可產生一17.5 kDa 的蛋白，其與ToMV鞘蛋白製備之血清有專一性反應。將pTCP之CP片段轉殖到Ti二位元體pBI121上，作煙草之轉型作用，在選擇性培養基培養6週生成轉基因煙草，包括ToMV局部病斑寄主Nicotiana tabacum cv. Havana六個株系之轉基因煙草、系統性寄主N. tabacum cv. Samsun九個株系，及Ntabacum cv. Xanthi五個株系之轉基因煙草。以西方墨點法及ELISA可偵測得到轉基因值物中有ToMV CP 的蛋白質產物，同時以PCR 亦可放大轉基因中含ToMV CP DNA 片段，經接種ToMV 1 μg/ml，此鞘蛋白轉基因煙草在局部病斑之煙草中，與非轉基因植物比較只產生其18 - 21 % 病斑數，而在系統性煙草中，接種ToMV0.5μg/ml，與非轉基因植物比較，其病徵延緩3-18天出現。本研究解析出台灣ToMV的鞘蛋白基因核苷酸序列，並了解其特性；且由轉型煙草之構築提供了一防治模式，可應用於台灣目前被ToMV為害相當嚴重之作物，如番茄、甜椒及辣椒等。

關鍵字

番茄嵌紋病毒 ； 鞘蛋白基因 ； 轉基因植物

並列摘要

Tomato mosaic virus (ToMV) is widespread and often epidemic in tomato and pepper crops in Taiwan. A cDNA clone pTCP was obtained from RT-PCR amplification of ToMV RNA of a virus isolate originated from tomato. The complete nucleotide sequence of the coat protein (CP) gene of ToMV was determined from subclones of restricition enzyme fragments of pTCP. The clone pTCP covered the entire CP gene that contained 690 nucleotides in length with a capacity encoding a protein of 158 amino acids. Comparison of the nucleic acid sequence of the CP gene of the Taiwan ToMV with that of the published Japan isolate showed that there were three nucleotides different and the amino acid sequences of the CP genes of both isolates arc identical, indicating that the Taiwan ToMV is almost identical to the Japan ToMV. In virro translation from the in vitro transcript generated from pTCP produced a protein with a M_r of 17.5 kDa which reacted specifically with the antibody to the ToMC CP. The ToMV CP fragment was digested from pTCP and subcloned in the Ti binary vector, and used for transformation of tobaccos, including the local lesion host of Nicoriana rabacum CV. Havana and the systemic hosts of N. rabacum cv. Samsun and cv. Xanthi. There were 20 transgenic tobacco lines regenerated, including 6 lines of Havana (T2, T3, T8, T11, T12 and T15), 5 lines Xanthi (X1, X2, X4, X5 and X7) and 9 lines of Samsun (S1, S2, S3, S6, S8, S9, S10, S11 and S12). The presence of the ToMV CP cDNA in the putative transgenic plants was confirmed by PCR using primers specific to the CP gene. A protein product similar to ToMV-CP was detected in the transgenic plants when they were analyzed by Western blotting and indirect ELISA. When the transgenic Havan a plants were inoculated with ToMV at 1 μg/ml. the numbers of local lesions were only 18-21% as compared with those on untransformed tobacco. In systemic hosts, symptom development was delayed 3- 18 days as compared with the control after they were inoculated with ToMV at 0.5 μg/ml. The construction of the ToMV CP-transgenic tobacco plant s resistant to ToMV infection provided a model system for control of diseases caused by ToMV in tomat o, sweet pepper and chili pepper in Taiwan.

並列關鍵字

Tomato mosaic virus ； coat protein ； transgenic plant