請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/22251
標題: PsbA或PsbO蛋白欠缺可誘導小球藻Chlorella sp. DT產氫&;#37238;
PsbA or PsbO suppression leads to hydrogenase induction in Chlorella sp. DT
作者: 林昕笛
Lin, Hsin-Di
關鍵字: Chlorella
hydrogenase
sulfur-deprived
anaerobic
transformation
HydA
PsbA
PsbO
RNA interference
knock down
出版社: 生命科學系所
摘要: PsbA (D1蛋白)是位在類囊體中的光合作用系統二(PSII)內的主要蛋白質之ㄧ; PsbO是位於類囊體內腔中PSII放氧複合體(OEC)的次單元蛋白之ㄧ。此二蛋白質皆與釋放氧氣的PSII穩定性有關。位在葉綠體基質的產氫&;#37238;(HydA),在無氧時可被誘導並且產生氫氣,但是在有氧環境時,其活性會受到抑制。 我們利用RNA干擾(RNAi)的方式使小球藻Chlorella sp. DT (DT)的psbA及psbO基因表現下降。當PsbA及PsbO 蛋白欠缺時,會阻礙PSII的組裝,進而抑制氧氣釋放及誘導HydA生成。 將帶有反義(antisense)基因片段antisense-psbA、antisense-psbO-a及antisense- psbO-b的小片段干擾RNA(siRNA)質體(pRPsbA、pRPsbO-a及pRPsbO-b)分別轉殖入小球藻DT細胞中。在轉殖株中,偵測到抗hygromycin基因(hpt),且在psbO轉殖株中觀測到cGFP的表現,得知siRNA質體成功地被轉殖到DT。在轉殖株中,發現其PsbA及PsbO的蛋白質含量較野生株為低,表示siRNA在DT轉殖株中發揮作用。我們得到了A1、A2、A5、Oa2、Oa11、Oa12、Ob2及Ob4這些PsbA或PsbO蛋白欠缺的轉殖株。在A2、Oa11、Oa12、及Ob4轉殖株中,更偵測到產氫&;#37238;基因(hydA)的轉錄以及HydA的表現。所以,在含硫的條件下,這些轉殖株中的HydA是可以被誘導出來的。
PsbA (D1 protein) is one of the core proteins of photosystem II (PSII) in thylakoid membrane; PsbO is one of the oxygen-evolving complex (OEC) subunits of PSII in the lumen side. Both concerned with the stability of PSII for O2 evolution. Hydrogenase (HydA), located at chloroplast stroma, is induced under anaerobic condition and capable of producing H2 but inhibited under aerobic condition. We attempted to knock down the psbA and psbO gene expression of Chlorella sp. DT (DT) by RNA interference as PsbA or PsbO supreesion would lead to the inhibition of O2-evolution and induction of HydA. The DT cells were transformed with short interference RNA (siRNA) plasmids pRPsbA, pRPsbO-a and pRPsbO-b which contained the antisense gene fragments of antisense-psbA, antisense-psbO-a and antisense-psbO-b, respectively. The observation of hpt fragments and cGFP in psbO transformants suggested that the siRNA plasmids had been successfully transformed into DT. PsbA and PsbO proteins were detected by western blotting. The low amount of PsbA or PsbO in transformants, as compared to wild-type, implied that siRNAs might function. The transcription of hydA and the expression of HydA in certain transformants suggested that HydA could be induced in these transformants under sulfur-supplied condition. In consequence, the PsbA or PsbO suppression transfomants of A2, Oa11, Oa12, and Ob4 were obtained. .
URI: http://hdl.handle.net/11455/22251
顯示於類別:生命科學系所

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