馬鈴薯 Y 病毒雙重表達載體之開發

Abstract

馬鈴薯 (Solanum tuberosum subsp. tuberosum) 是世界上重要的非穀類作物。馬鈴薯產業在台灣連年成長，目前也培育出多新品種，如台農一號、種苗二號和台農四號。為了提升馬鈴薯的產量及增強其抗病與抗逆境能力，深入探索參與農藝性狀改良的關鍵基因至關重要。一般分析基因的方法依靠轉基因技術，然而常見的馬鈴薯品種為四倍體，再加上其自交不親和的特性使得轉基因技術應用於馬鈴薯的效率低且耗時耗力。為了克服這些障礙，開發病毒載體以進行基因功能性分析為當務之急。反向遺傳學技術，如病毒誘導基因靜默 (virus-induce gene silencing, VIGS) 和病毒介導的過表達 (virus-mediated protein overexpression ,VOX)，能透過調控基因表現快速影響植物表型變化，因而成為研究馬鈴薯基因功能的重要工具。此技術可應用於研究植物的抗病蛋白與病原的相互作用與作用機制。本研究選擇馬鈴薯 Y 病毒 (potato virus Y, PVY) 做為病毒載體開發對象，PVY 編碼一多聚蛋白，並經由病毒蛋白酶裂解成多個成熟的蛋白。此策略的優點為可以同時將多個外源序列插入 PVY 基因體中進行表現，使其與病毒多蛋白共同轉譯後經蛋白酶裂解，實現多個外源序列之同步表現。為建立以 PVY 為基礎之病毒載體，本研究首先自雲林分離獲得弱毒株 PVY (PVY-96NTNa)，與強毒株 PVY-33N-Wi 相比，PVY-96NTNa 不僅未引起明顯病徵，亦表現出更快速且更高的病毒累積量。此外，進一步比較兩分離株之 P1/HC-Pro 及 VPg 蛋白之 RNA 靜默抑制能力 （RNA silencing suppressor, RSS）後發現，PVY-96NTNa 之 RSS 能力並未顯著低於 PVY-33N-Wi，揭示其致病力削弱可能與 RSS 能力無直接關聯。接續地，本研究成功將 PVY-96NTNa 之全長 cDNA 構築為感染性選殖株 pPVYm。為進一步改造pPVYm 成為雙重表達病毒載體 pPVY-dual，分別於 P1/HC-Pro 與 NIb/CP 交界處插入兩個限制酶切位 BstZ17I 與 SnaBI，後續將類胡蘿蔔素酶 （phytoene desaturase, PDS） 之部分基因片段及綠色螢光蛋白 （green fluorescent protein, GFP） 之完整編碼序列插入雙基因插入框中，以評估其基因靜默與外源蛋白表現之能力。結果顯示，pPVY-dual 能有效表現 GFP，且其 GFP 插入片段於後代植株中仍可穩定存在。然而，pPVY-dual 並未成功誘導 PDS 基因靜默。儘管目前尚未建立高效率之 VIGS 系統，但 pPVY-dual 具備低致病性、高複製能力、遺傳穩定及雙插入位點設計等特性，顯示其具潛力發展為應用於馬鈴薯功能基因體學研究之 PVY 病毒載體平台。

Potato (Solanum tuberosum subsp. tuberosum) is one of the world's most important non-cereal crops. The potato industry in Taiwan has seen continuous growth in recent years. To improve potato traits such as yield and resistance to biotic and abiotic stresses, identifying and characterizing key genes involved in these processes is essential for effective trait enhancement. Traditional methods for gene functional analysis rely on transgenic technologies; however, most cultivated potato varieties are tetraploid and exhibit self-incompatibility, making the application of transgenic technologies in potatoes inefficient and time-consuming. To overcome these obstacles, it is important to develop virus vectors for functional gene analysis including virus-induced gene silencing, (VIGS) and virus-mediated protein overexpression (VOX). In this study, we select potato virus Y (PVY) for development of viral vector, since PVY encodes one large polyprotein and further cut by proteases into many mature proteins. The advantage of using this strategy is that multiple foreign sequences can be inserted into the PVY genome to encode a polyprotein with foreign sequences and then be cleaved by proteases leading to simultaneous expression of multiple foreign sequences. To develop a PVY-based viral vector, we first identified a mild strain of PVY (PVY-96NTNa) isolated from Yunlin. Compared to the severe strain PVY-33N-Wi, PVY-96NTNa accumulates more rapidly and to higher levels, yet causes no visible symptoms. Moreover, the RNA silencing suppressor (RSS) activities of P1/HC-Pro and VPg in PVY-96NTNa are not significantly different from those in PVY-33N-Wi, suggesting that the attenuated pathogenicity of PVY-96NTNa may not be directly linked to its RSS capacity. The full-length cDNA of the PVY-96NTNa isolate was cloned to generate an infectious clone, designated pPVYm. To further engineer pPVYm as the dual-expression vector, pPVY-dual, two restriction sites, BstZ17I and SnaBI, were subsequently introduced into the PVY-96 genome at the P1/HC-Pro and NIb/CP junctions, respectively. Subsequently, a partial fragment of the PHYTOENE DESATURASE (PDS) gene and the coding sequence of green fluorescent protein (GFP) were inserted into the pPVY-dual vector to assess its gene silencing and expression capabilities, respectively. Our results showed that pPVY-dual can successfully express GFP and the GFP cassette remained intact in the next host generation. By contrast, pPVY-dual failed to perform VIGS. Although high-efficiency VIGS has not yet been achieved, the low symptom severity, high replication capacity, genetic stability, and dual-insertion cassette design of pPVY-dual provide a robust platform for the future development of PVY-based tools in functional genomics in potato.