Construction of in Vitro and in Vivo Infectious Transcripts of a Taiwan Strain of Zucchini yellow mosaic virus

臺灣矮南瓜黃化嵌紋病毒生體外及生體內具感染能力轉錄體之構築

林詩舜(Shih-Shun Lin) ； 侯豐男(Roger F.Hou) ； 葉錫東(Shyi-Dong Yeh)

《Botanical Bulletin of Academia Sinica》 43卷4期 (2002/10) Pp. 261-269

https://doi.org/10.7016/BBAS.200210.0261

摘要

矮南瓜黃化嵌紋病毒臺灣分離株(ZYMV TW-TN3)之全長基因體選殖株經由五個互補DNA片段之選殖株連接完成。將全長基因體構築成由T7啟動子所調控的pTT7ZYMV2-5質體。所產生之生體外具感染力轉錄體具有TW-TN3全長病毒核酸序列(9591 nt)並且其5＇及3＇端分別額外產生一個鳥糞嘌呤與5個poly(A)。此外，pT7ZYMV2-5亦被改造成由花耶菜嵌紋病毒3＇端含有一個nos終止子。由pT7ZYMV2-5所生成的RNA轉錄體與所純化的p35SZYMV2-26質體DNA可以分別以機械接種及粒子槍接種的方式感染矮南瓜植物。經由生體外與生體內具感染力轉錄載體所接種的矮南瓜植物在接種後四到六天即可以觀察到系統性病徵。此外兩者亦可以利用機械接種的方式感染單斑寄主奎藜植物。經由感染力測試、病徵型態觀察及免疫金標定電子顯微鏡分析顯示，由pT7ZYMV2-5及p35SZYMV2-26質體所產生的TW-TN3生體內與生體外轉錄載體具備感染植物的能力。此種具備感染力的生體外或生體內轉錄體之構築對ZYMV TW-TN3的分子研究提供了一個重要基石。

關鍵字

ZYMV臺灣分離株 ； 生體內 ； 生體外 ； 具感染力轉錄體

並列摘要

The full-length cDNA of a Taiwan strain of Zucchini yellow mosaic virus (ZYMV TW-TN3) was constructed from five overlapping cDNA clones downstream from the bacteriophage T7 promoter in plasmid pT7ZYMV2-5. The plasmid was able to generate an in vitro transcript corresponding to TW-TN3 (9591 nt) with one extra guanosine residue at the 5’ terminus and a poly(A)95 tract at the 3’ end. In addition, pT7ZYMV2-5 was used for the construction of p35SZYMV2-26 that contained the full-length cDNA of TW-TN3 with a Cauliflower mosaic virus (CaMV) 35S promoter and a nopaline synthase (nos) terminator. The capped in vitro transcript generated from pT7ZYMV2-5 and the purified DNA of p35SZYMV2-26 were introduced into zucchini squash plants by mechanical inoculation and particle bombardment, respectively. Both in vitro and in vivo transcripts induced systemic symptoms on zucchini squash 4 to 6 days after inoculation. In addition, both transcripts also induced local lesions on plants of Chenopodium quinoa by mechanical inoculation. The results of infectivity assay, symptomatology, and serologically specific electron microscopy indicated that the in vitro and in vivo TW-TN3 transcripts derived from pT7ZYMV2-5 and p35SZYMV2-26, respectively, are infectious. The ability to generate biologically functional transcripts from the constructed eDNA clones is a significant step for molecular analyses of TW-TN3.

並列關鍵字

In vitro ； In vivo ； Infectious transcripts ； ZYMV TW-TN3.