以表現輔助蛋白提升農桿菌的植物轉殖效率

Abstract

農桿菌常被廣泛應用於植物基因轉殖技術中，因其操作簡易，可使目標基因單一且完整的插入染色體組中，並且不易引發基因靜默 (gene silencing)反應，因此，近30年來以此法進行轉殖成功的植物超過百種，然而仍有許多植物難以進行基因轉殖，其中包括蘭花、百合等重要花卉作物，故如何提升植物轉殖效率仍舊是極為重要的課題。農桿菌轉殖法中的T-DNA傳送過程需要許多輔助蛋白 (accessory protein)參與，包括來自農桿菌或植物的蛋白質，例如BTI、VIP1、Ku80、H2A等。許多文獻曾指出經基因轉殖使大量表現輔助蛋白之擬南芥，其T1世代植株被再度轉殖的效率大幅提升，暗示輔助蛋白可能扮演提升轉殖效率的角色。

由於上述研究材料均僅侷限於雙子葉之模式植物-擬南芥，因此本實驗選用可轉譯出輔助蛋白的水稻同源基因，稱之為促轉基因 (enhance transformation genes，簡稱為ET gene)，構築於植物表現載體上，分別對文心蘭與擬南芥進行轉殖實驗，希望在單、雙子葉植物中均測試促轉基因的效果，並於T0世代(即野生株)即進行評估，以達到利用促轉基因提升目標基因轉殖效率的實用目的。實際作法上乃利用分別帶有促轉基因或GUS報導基因的兩種農桿菌，進行共同轉殖 (co-transformation)，數日後藉由偵測短暫性表現的GUS蛋白活性研判促轉基因是否可提升轉殖效率。循此想法，首先需測試雙農桿菌的共轉殖效率，以分別帶有紅、綠螢光蛋白的兩株農桿菌共同感染文心蘭，其後製成原生質體 (protoplast)觀察，發現農桿菌共同轉殖單一細胞的效率高達90%。故以空植物表現載體為控制組，以促轉基因為實驗組，分別與帶有GUS報導基因的農桿菌進行共同感染，發現GUS報導基因於文心蘭中的表現活性極低，難以用於進行定量評估，故將改以強啟動子或病毒的抑制基因 (viral suppressor gene)提升GUS報導基因的基礎表現量，以真正評估促轉基因於文心蘭轉殖的效果。另一方面，擬南芥的測試顯示共轉殖ET2、ET6與ET9促轉基因可提升暫時性GUS基因的表現量 (p< 0.05，Student’s t-test)，暗示T-DNA被傳送入核的頻率上升。然而，於永久性轉殖實驗，發現ET7可提升2.5倍的轉殖效率(p< 0.05，Student’s t-test)，經聚合酶鏈鎖反應及南方墨點法，不論促轉基因同時插入染色體的頻率或GUS報導基因的嵌入複本數均與控制組無太大差異。暗示ET7促轉基因可提升GUS報導基因永久性轉殖效率，但不會提升嵌入的複本數。

Agrobacteria-mediated transformation of higher plants is a well-known and powerful tool for T-DNA delivery to plant cells. The method results in mostly single or low-copy integration of full-length transgene, and is less likely to trigger gene silencing than gene-gun mediated method. Various technical modifications, either on Agrobacteria or plant side, were employed to improve the transformation efficiency ever since its invention on 1983. However, many plant species or cultivars are still granted as “recalcitrant” that barely give rise to regenerable and non-chimeric transgenic lines. Therefore, it is still critical to improve efficiency of this technique. The long journey for T-DNA transferring till the final destination not only requires bacteria proteins but also is aided by various plant factors, including BTI, VIP1, Ku80, H2A, etc. Interestingly, T1 progenies of transgenic Arabidopsis overproducing several accessory proteins were found to exhibit higher transformation efficiency, suggesting roles of the accessory proteins in “enhance transformation”, designed as “ET genes” in this study. To test if this condition could be employed at the T0 stage, so to enhance delivery of target gene directly, we use co-transformation strategy with two T-DNA harbored in two Agrobacteria. One T-DNA carries simply the ET gene ortholog from rice and the other T-DNA contains GUS reporter gene together with hygromycin selection marker. Oncidium and Arabidopsis were chosen as the organisms to test. To evaluate the efficiency of Agrobacteria-mediated co-transformation in Oncidium, protoplast was prepared after transformation and observed for frequency of co-presence of GFP and RFP proteins within single cell. Surprisingly, the co-transformation efficiency reached ~90%, indicates a feasibility of our strategy. Unfortunately, the expression level of GUS protein in protocorm-like bodies (PLBs) of Oncidium was too low for quantitative assays, even strong promoter and anti-silencing viral suppressors were employed for boosting gene expressions. On the other hand, using Arabidopsis root segments as materials did identify three ET genes, ET2, ET6 and ET9, in enhancement of transient transformations to a significant level (p<0.05 in Student's t-test). Moreover, floral-dip method for producing transgenic Arabidopsis was improved by the co-presence of ET7 to 2.5-fold. The co-integration frequency of ET gene and copy number of transgene were not particularly elevated in the transgenic lines examined, suggesting a usage of ET7 to increase the transformation frequency without a deleterious effect.