綠竹超氧歧化酶基因之選殖及生化特性之研究

Molecular Cloning and Biochemical Characterization of Green Bamboo (Bambusa oldhamii) MnSOD and CuZnSOD

吳宗翰(Tsung-Han Wu)

指導教授 ： 靳宗洛

國立臺灣大學/生命科學院/植物科學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.02788

摘要

綠竹超氧歧化酶的同功酶活性，主要由銅/鋅超氧歧化酶及錳超氧歧化酶所構成。在原態膠體上進行活性染色，可分出七個銅/鋅超氧歧化酶活性條帶及一個錳超氧歧化酶活性條帶，相較於其他植物，綠竹具有更多的銅/鋅超氧歧化酶活性條帶。在綠竹的粗萃取液中加入濃度為0.05至0.2 mM的銅離子或是以8 M尿素將之變性再還原，皆未造成該七個銅/鋅超氧歧化酶活性條帶分布位置的改變。表示這些銅/鋅超氧歧化酶活性條帶並非因攜帶的銅離子數目不同，或是由不同數目的次單位體聚合而成。經由南方氏轉印分析發現：綠竹銅/鋅超氧歧化酶及錳超氧歧化酶的基因數目約有四至五個。這些結果顯示綠竹具有許多超氧歧化酶基因，並且具有許多銅/鋅超氧歧化酶的同功酶活性。推測綠竹可能具有更複雜的抗氧化系統以及調控機制，來參與其在氧化逆境下所表現出的一些特性，例如:生長快速。
經由比對其他單子葉植物的錳超氧歧化酶及銅/鋅超氧歧化酶cDNA序列，設計保守引子對進行3’-RACE 及 5’-RACE，得到兩條錳超氧歧化酶coding region序列，經軟體分析，發現其轉譯之蛋白質序列之N端，皆具有可能之粒線體transit peptide，故推測此錳超氧歧化酶可能會進入粒線體並清除超氧。將得到之錳超氧歧化酶與其他單子葉植物的錳超氧歧化酶進行比對，其相似度在75~89%之間。另外，銅/鋅超氧歧化酶部分，也得到四條coding region序列，經軟體分析，推測此四條cDNA皆為細胞質型銅/鋅超氧歧化酶。此四條銅/鋅超氧歧化酶和其他單子葉植物銅/鋅超氧歧化酶的相似度，在81~95%之間。
進一步將銅/鋅超氧歧化酶及錳超氧歧化酶的coding region序列，接入pGEX-6P-1表現載體中，使之在大腸桿菌中大量表現銅/鋅超氧歧化酶及錳超氧歧化酶的重組蛋白質，並利用親合性層析法進行GST-BoCuZnSOD及GST-BoMnSOD重組蛋白純化。純化之GST-BoMnSOD及GST-BoCuZnSOD皆具有超氧歧化酶活性，同時在鹼性pH值下或是在低於60℃下呈現穩定的活性，並在室溫下放置三天仍保有良好的酵素活性。
本論文所得到的結果:在大腸桿菌裡表現的綠竹超氧歧化酶重組蛋白，可在鹼性的環境、高溫，以及長時間在室溫下，保有良好的活性，這些特性，將來或許可開發利用於日常生活用品中，例如皮膚表面自由基的清除劑。

關鍵字

綠竹 ； 超氧歧化酶

並列摘要

The total superoxide dismutase (SOD) isozymes activity in green bamboo were analyzed, at least seven isoforms of CuZnSOD and one MnSOD were identified. The pattern of SOD activities were not affected when the crude protein extracts prepared from bamboo leaves were incubated with 0.05-0.2 mM copper ions, 8 M urea or dialyzed after the urea treatment. The results revealed that the seven CuZnSOD activity bands were not caused by the different number of copper ions in the proteins or different number of subunits in the native form of CuZnSOD. Southern blotting analyses revealed that there were at least four to five CuZnSOD and MnSOD genes in green bamboo. The abundance of SOD genes and the complexity of CuZnSOD isoforms suggested that the green bamboo might contained a more complex regulation system for the detoxification of reactive oxygen species (ROS)to cope with the . oxidative stresses.
The published MnSOD and CuZnSOD cDNA sequences in monocots were aligned and the degenerated primers were designed according to the conserved regions. These primers were used for RT-PCR, 5’- and 3’-RACE experiments and two BoMnSOD cDNA were cloned from the green bamboo. The deduced amino acid sequences of the two BoMnSOD consist of 231 amino acids, including a putative mitochondrial transit peptide (27 a.a.) in the N-terminus and the essential domains of MnSOD. The identity of the two BoMnSOD genes and other MnSODs genes monocots was 75-89%. Four BoCuZnSOD cDNA were also cloned. The deduced amino acid sequences of the BoCuZnSOD consists of 152 amino acids and it is predicted as a cytosolic form protein. These BoCuZnSOD shares 81-95% identity with other monocot CuZnSODs genes. The coding region of BoMnSOD and BoCuZnSOD were cloned into the pGEX-6P-1 expression vector. the GST-fusion proteins were expressed in the Escherichia coli and purified by the GST affinity column. The purified GST-BoCuZnSOD and GST-BoMnSOD remained the SOD activity. Both fusion proteins were stable at alkaline pH and declined to 10% after incubation at 60°C for 20 minutes. The activity of the fusion GST-BoCuZnSOD and GST-BoMnSOD were also stable under room temperature for 3 days.
BoMnSOD and BoCuZnSOD cDNA from green bamboo can be expressed in prokaryote (E. coli) and remain stable activity under a broad range of pH, higher temperature, also very stable in the room temperature.
These properties are beneficial for applications in commercial, such as in cosmetics for skin protection of defending unesthetic effects caused by oxygen-containing free radicals.

並列關鍵字

green bamboo ； superoxide dismutase

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