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Cross-protection against Cucumber green mottle mosaic virus using mild strain Zucchini yellow mosaic virus carrying the coat protein and partial replicase gene of CGMMV
|摘要:||胡瓜綠斑嵌紋病毒(Cucumber green mottle mosaic virus, CGMMV)為Tobamovirus屬，其基因型態與菸草嵌紋病毒相似。此病毒寄主範圍狹窄，主要感染瓜類作物，在瓜類上產生嚴重病害，造成經濟上嚴重的損失。在本實驗室中矮南瓜黃化嵌紋病毒（Zucchini yellow mosaic virus, ZYMV）已被發展成在生體內具感染力的病毒載體。此外，本實驗室亦將此病毒載體之協同性蛋白（HC-Pro）上的第180個及第396個胺基酸進行突變（Arg180àIle, Glu396à Asn）。突變後的病毒菌株具較低的毒性，亦可作為用以表現外源蛋白之病毒載體且具有交互保護的能力。為了同時防治胡瓜綠斑嵌紋病毒及矮南瓜黃化嵌紋病毒在瓜類植物上造成的危害，本實驗將CGMMV的186 kDa複製酶3'端處的基因片段以及CGMMV之鞘蛋白基因導入弱毒性的矮南瓜黃化嵌紋病毒載體內。導入的部位為病毒載體的P1及HC-Pro蛋白基因間，或者是NIb及CP蛋白基因間。此外，為了增加重組病毒的穩定性，186 kDa複製酶3'端處的基因片段被分成前、後兩個片段。這兩片段同樣地被分別導入弱毒性病毒的P1及HC-Pro蛋白基因間，或是NIb 及CP蛋白基因間。結果顯示當外源的CGMMV基因片段導入於NIb 及CP蛋白基因間時，使得重組體變得不穩定，導致外源DNA片段容易被剔除。而在交互保護的實驗中，於刺角瓜上接種這些輕症型重組病毒仍可使植物免於重症型ZYMV病毒的感染。然而，在對抗CGMMV的感染上，攜帶CGMMV RdRp片段的重組病毒只能提供部分的保護效果，而帶有CGMMV 鞘蛋白的輕症型病毒載體則完全不具交互保護的能力。|
Full-length cDNA clone of the severe strain Zucchini yellow mosaic virus (ZYMV TW-TN3) driven by a Cauliflower mosaic virus 35S promoter was preciously engineered as an in vivo infectious viral vector in our laboratory. The mild strain ZYMVAC vector, with mutations at Ile180 and Asn396 of the HC-Pro protein, was also developed and conferred excellent cross-protection ability against severe strain ZYMV infections. In this investigation, the reathrough portion of 186 kDa protein and coat protein (CP) genes of Cucumber green mottle mosaic virus (CGMMV), an important virus limiting cucurbits production, were used for further tests for cross-protection against the unrelated CGMMV. The readthrough portion of 186 kDa protein and the CP genes of CGMMV were inserted into the ZYMVAC vector between the P1 and HC-Pro or between NIb and CP, respectively. For improving the stability of ZYMVAC recombinants, the 1.5 kb fragment of the readthorugh portion of 186 kb protein was further divided into two portions, N'-terminal and C'-terminal. The N'-terminal and C'-terminal were also inserted into mild strain vector between the P1 and HC-Pro or between NIb and CP, separately. The resulted ZYMVAC recombinants carrying the heterologous ORF of CGMMV between P1 and HC-Pro were tested for their cross-protection effectiveness against CGMMV. All ZYMVAC recombinants remained the ability to confer the high degree of cross-protection against ZYMV. However, these recombinants only conferred partial protection against CGMMV, as reflected in delay of the development of the severe symptoms of the challenge virus, and the protection was overcome more quickly when using the ZYMVAC vector carrying the CGMMV CP as the protective virus. The reasons of inability of these recombinants to confer complete protection against CGMMV are discussed.
|Appears in Collections:||植物病理學系|
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