共同表現GUS與GFP於菸草毛狀根

Abstract

為了建立以根毛農桿菌介導轉形多基因的方法，我們將報導基因各建構於不同的雙元質體，分別由兩菌體攜帶(2AR)或由單一菌體攜帶(2BV),另外也設計於同一雙元質體中將兩不同報導基因置於同一T-DNA中(1TD)或是不同的T-DNA中(2TD)對菸草進行感染與轉形研究。比較此四種方法之誘導率、DNA轉形效率、以及異源蛋白GUS與GFP的表現量，結果顯示，四種方法的毛狀根誘導率皆達到九成以上，且無異於野生型根毛農桿菌 (Agrobacterium rhizogenes) 的誘導率。2AR、2BV、1TD、2TD的轉形效率分別為65.4%、40%、78.6%、82.1%，以1TD與2TD的轉形效率較佳。另外，結果顯示此四種策略均可在菸草毛狀根中共表現GUS與GFP，。在轉形毛狀根的表現方面， 2AR、2BV、1TD、2TD的GUS活性分別介於0~259.1、0.2~698.9、0.7~112.3、0.6~698.9 nmole MU min-1 mg protein-1；在GFP表現量方面，則分別介於5.5~6333.9、0.4~8758.7、1.3~1923.5、0.7~8459.3 ng mg-1 protein。綜合來看，1TD與2TD是較好的共轉形及共表現組，但四種轉形策略皆可對植物進行多基因的轉形。

To establish a platform of Agrobacterium rhizogenes-mediated multiple-gene transformation, we proposed and evaluated different strategies in this study. Two reporter genes are carried by two separate plasmids within different transformed A. rhizogenes (2AR) and within one transformed A. rhizogenes (2BV). Two reporter genes are also constructed in one T-DNA (1TD) and in different T-DNA (2TD) in one binary vector. In the study, we compare the hairy root inducing rate, genes co-transforming rate and the expression of reporter genes-GUS and GFP. Hairy root induction rate of the four groups were all over 90%, without difference from wild type A. rhizogenes. The transformation efficiency of 2AR, 2BV, 1TD and 2TD was 65.4%, 40%, 78.6%, and 82.1%, respectively. Our results showed that all of the four strategies can co-express GUS and GFP in tobacco hairy roots. The GUS expression of 2AR, 2BV, 1TD and 2TD ranged from 0 to 259.1, from 0.2 to 698.9, from 0.7 to 112.3, and from 0.6 to 698.1 nmol MU min-1 mg-1 protein, respectively. Meanwhile, the transgenic hairy roots expressed GFP ranging from 5.5 to 6333.9, from 0.4 to 8758.7, from 1.3 to 1923.5, and from 0.7 to 8459.3 ng mg-1 protein, respectively. 1TD and 2TD were better strategies to gain co-expression hairy roots, while all of the four strategies were able to transform multiple genes into plants.