- 學位論文
利用病毒誘導基因沉寂系統探討銅逆境下菸草脯胺酸之生成及代謝
The studies of proline metabolism in Nicotiana benthamiana under copper stress using virus-induced gene silencing system
摘要
植生復育法乃利用植物吸收污染地之重金屬以復育土壤。但植物於過量重金屬環境下，會進行生理調節機制以抑制重金屬進入植體，因此，導致效果不彰。而脯胺酸的增加，可能是其中的一種阻擋機制，但是脯胺酸的增加是否與重金屬進入植體有關，尚無定論。若能改變脯胺酸的含量，或許能使重金屬的進入植體不受阻擋，而有助於植生復育之效能提高。本研究目的，在於探討能否利用病毒誘導基因沉寂 (VIGS) 系統調控菸草脯胺酸的含量，進而瞭解菸草植體內的銅含量與脯胺酸變化的關係，藉此評估此系統應用於植生復育之效能。脯胺酸的合成路徑主要是透過兩個關鍵酵素Δ1-pyrroline-5-carboxylate synthetase (P5CS) 與ornithine -δ-aminotransferase (OAT)；而脯胺酸的代謝則主要是透過proline dehydrogenase (PDH)。本研究利用VIGS技術，於六週大的菸草進行接種，分別或同時降低上述基因之表現量。並於接種28天後，給予5 mM CuSO4處理六天。結果發現，抑制OAT基因會使菸草地上部植體乾重的銅含量增加1.2倍，而抑制P5CS基因亦增加為1.3倍，但同時抑制OAT與P5CS基因時，銅含量反而降為0.7倍。透過植物型態上的觀測，發現抑制OAT基因，在地上部方面會促使菸草側芽數增加，亦使菸草根部鬚根鮮重增加，而主根鮮重則降低。藉以推論，抑制OAT基因，會加速菸草發展鬚根，進一步促使銅含量提高。但抑制P5CS基因時，並無此現象。本研究也發現，抑制代謝相關基因PDH亦會使銅含量增加為2.5倍。由於抑制P5CS或PDH基因均可使脯胺酸的利用降低，顯示銅逆境下脯胺酸的增加阻擋銅進入植體之機制，可能與脯胺酸含量無關而是與脯胺酸的利用有關。此外，同時抑制OAT、P5CS及PDH基因，銅含量則增加為1.6倍，此結果介於單獨抑制OAT、P5CS基因與單獨抑制PDH基因之間，顯示同時抑制合成及代謝相關基因具有相互抵抗的作用，降低其復育效能。總言，利用病毒誘導基因沉寂系統，抑制菸草脯胺酸的合成與代謝，具有提高植生復育效能之潛力。
並列摘要
Phytoremediation is one of soil remediation strategies that remove heavy metal contaminants with plants. Under excess heavy metal condition, plants tend to initiate physiological adjustments to reduce the entry of heavy metal and result in the decrease of phytoremediation efficiency. The accumulation of proline was considered to be one of the adjustments that block the entering process. It is hypothesis that the inhibition of proline accumulation in plants could benefit the application of phytoremediation by decreasing the blocking mechanism, although there’s no direct evidence showed the accumulation of proline can block the entering of heavy metal. This study using virus induced gene silencing (VIGS) system in regulating the proline content was try to reveal the relationship between the variation of proline content and the heavy metal content in tobacco, and to assess the possibility of applying this system on phytoremediation. The key enzymes of two proline synthesis pathways are Δ1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine -δ-aminotransferase (OAT). The key enzyme of proline catabolism pathway is proline dehydrogenase (PDH). Six weeks old tobacco (Nicotiana benthamiana) plants were subjected for the application of VIGS to knock down the expression of proline metabolism related genes. After 28 days of inoculation, the plants were treated with 5 mM CuSO4 for six days. The results showed that the suppression of OAT gene enhanced tobacco copper content up to 1.2 fold, and the suppression of P5CS gene enhanced up to 1.3 fold. However, when both OAT and P5CS genes were suppressed the copper content decreased to 0.7 fold. In addition, the suppression of OAT gene increased the number of tobacco auxiliary buds, the fresh weight of fibrous root but decrease the fresh weight of tapper root. It is suggested that OAT suppression induced increasing of fibrous root might benefit the absorption of copper. Surprisingly, the suppression of PDH gene also enhanced copper content to 2.5 fold. Since both of the suppression on P5CS and PDH down regulated copper entry, the mechanism of blocking copper entry could be related to the utilization of proline instate of the proline level only. In addition, the result on the combined silencing of all proline metabolism related genes showed the amount of copper content was between those from the suppressed of proline synthesis and catabolism enzyme indicating that the silencing of proline metabolism relatived genes shows antagonism. In conclusion, the application of VIGS system to silence proline synthesis and catabolism relative genes has the potential to enhance the efficiency of phytoremediation.