以色澤蛋白作為微細藻基因轉殖之新穎篩選標記

Abstract

擬球藻為水產養殖產業中的重要光合自營藻類，常用於魚幼苗之活餌飼料。本實驗室於2009年時，已先行研究利用具有抗菌能力之牛乳鐵蛋白(LFB)，融合紅色螢光蛋白(DsRed)此報導基因作為篩選標記，選殖含牛乳鐵蛋白之擬球藻轉殖藻株。然而，利用紅色螢光蛋白選殖時，總共需時約兩個月才可進行篩選，且此紅螢光訊號亦會受到轉殖細胞所含有的內生性紅螢光干擾，進而影響選殖結果，因此本研究決定重新選擇新的篩選標記解決上述紅螢光蛋白作為篩選標記之缺點，使用了從地毯海葵(Stichodacyla haddoni)中，所分離出的紫色色澤蛋白（shCP），作為新一代的篩選標記，用以篩選擬球藻轉殖藻株。此研究中我們利用熱誘導型啟動子開啟下游shCP cDNA紫色色澤蛋白之表現，由於擬球藻轉殖藻株所表達之外源紫色色澤蛋白會與其內生性綠色之葉綠體彼此參雜，因而造成轉殖藻株呈現與綠色野生型藻株迥然不同之深褐色色澤。本實驗研究中我們證明shCP色澤蛋白為相當理想、快速，成本低廉的篩選標記，藉由其色澤特性，可有效地幫助研究人員以肉眼辨識之方式辨認並分離出轉殖藻株。此外，我們亦利用shCP此篩選標記建構出一新表現質體，此質體將六段重複之乳鐵蛋白序列與shCP篩選標記接合，並轉殖至擬球藻藻株中，於三個星期後成功地利用shCP篩選標記，篩選並分離出含有六段重複之乳鐵蛋白序列與shCP融合序列之轉殖藻株，後續實驗中我們亦證明該轉殖藻株具有抗微生物活性(antimicrobial activities)，證明shCP篩選標記不會影響乳鐵蛋白此功能性蛋白之功能，且shCP以及LFB為生物可分解蛋白(biodegradable)，具有環境生物安全性，因此可降低含抗菌基因之微生物之生成，並減少抗菌基因擴散到環境中，進而降低對環境生態所帶來之影響、衝擊。

Nannochloropsis oculata is an essential photoautotrophic microalgae used commonly as live feed for fish larvae in aquaculture. Previously we have been developed and isolated a transgenic line of N. oculata harboring antimicrobial peptide such as bovine lactoferricin (LFB) fused a red fluorescent protein (DsRed) served as selection marker. However, it takes two months to observe the appearance of DsRed reporter and DsRed is disturbed by the endogenous fluorescence of the host chloroplasts. Moreover, the fluorescent microscope is required to be equipped. To solve these disadvantages for screening the genetically engineered microalgae, new selection marker is needed. In this study, we used the purple chromoprotein (CP) isolated from Stichodacyla haddoni to serve as an effective selection marker to screen the transgenic colonies of N. oculata. The expression of S. haddoni purple chromoprotein (shCP) cDNA was controlled by an inducible promoter. Since the expressed shCP was mixed with the endogenous green chloroplasts of N. oculata, dark brown color was display in transformants. We demonstrated that shCP reporter is a reliable, rapid and cheap approach which enables to identify and isolate transgenic cells. Furthermore, we also developed an expression plasmid, in which shCP reporter gene was fused with six repeats of LFB cDNA, and transferred into microalgae. After we successfully screened and isolated a transgenic line of N. oculata harboring shCP fused with LFB within three weeks, we proved that this variety had antimicrobial activity, resulting in greatly increasing the survival rate of fish infected with bacterial pathogens. Using transgenic N. oculata containing LFB should reduce the antibiotic usage in aquaculture industry. Importantly, since shCP and LFB proteins are biodegradable and originates from marine organism, it is considered as a biological and environmental safety, which should also substantially reduce the public concern related to generate antimicrobial-resistant bacteria and resistance genes to the environment and thus bring ecology impact.