Antisense and RNAi Expression for a Chloroplastic Superoxide Dismutase Gene in Trausgenic Plants

從番茄中克隆而得的葉綠體銅鋅超氧化物歧化酶(Cu/Zn-SOD)基因通過轉基因技術導入煙草中。雙鏈RNA (dsRNA)和反義RNA結構能特異地抑制轉基因煙草葉綠體Cu/Zn-SOD的表達，其機理是內源葉綠體Cu/ZnSOD基因成為了髮卡RNA (hpRNA)和反義RNA的靶子而表現為沉默。與野生型煙草相比，不僅SOD整體活性下降，而且原應存在於葉綠體中的Cu/Zn-SOD同功酶條帶完全消失。在鹽和聚乙二醇(PEG)脅迫中，兩種轉基因煙草胞質Cu/ZnSOD活性明顯上升，而PEG的預處理使植株的SOD活性上升早於未進行預處理的植株。結果說明，通過雙鏈RNA和反義RNA結構能有效的抑制目的基因的表達，但由於同功酶的存在，轉基因植株在脅迫條件下仍能保持一定的總SOD活性，且其他SOD同功酶活性能補償葉綠體Cu/Zn-SOD的缺失。總而言之，RNAi技術對於揭示葉綠體Cu/ZnSOD的功能有極大的幫助，而且發現在脅迫條件下植物與Cu/Zn-SOD相關的抗氧化過程存在胞質同功酶的交替途徑，同時也為生煙草中進行反義抑制的操作提供了技術支援。

關鍵字

超氧化物歧化酶；雙鏈RNA ；轉基因煙草；鹽脅迫

並列摘要

Abstract. The cDNA of tomato chloroplastic Cu/Zn-superoxide dismutase was used to construct transgenic tobacco (Nicotiana plumbaginifolia). It was found that the gene expression of Cu/Zn-superoxide dismutase can constitutively be reduced in the transgenic tobacco plants because of double-stranded (dsRNA) expressed in the form of intron-spliced hairpin structures and antisense suppression. Furthermore, the endogenous chloroplastic Cu/Zn-superoxide dismutase gene in tobacco was established as a target for silencing due to the operation of hpRNA and antisense RNA constructs. However, under salt and PEG-induced stress, the cytosolic Cu/Zn-superoxide dismutase activity in transformed plants obviously increased. At the same time, the PEG pretreatment was able to promote tolerance of the transgenic plants to the salt stress. These results indicated that the use of hpRNA and anti-sense was able to successfully knockout the transcript encoding a chloroplast superoxidase dismutase. The null transformed plants grown under stress produced or retained other superoxide dismutase to compensate for the loss of the chloroplast. Consequently, hpRNA constructs would be helpful in discovering and validating the endogenous chloroplastic Cu/Zn-superoxide disinutase gene, and to prove the cytosolic alternative pathway of plant antioxidation associated with the function of Cu/Zn-superoxide dismutase gene under an unfavorable environment. Meanwhile, the experiment will provide an important technique for the antisensing strategies operating in tobacco.

並列關鍵字

Double-stranded RNA ； Salt stress ； Superoxide dismutases ； Transgenic tobacco

國際替代計量

Antisense and RNAi Expression for a Chloroplastic Superoxide Dismutase Gene in Trausgenic Plants

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