摘要:在石油短缺與溫室效應加劇的今天,微藻產油已為大家重視,也是此次國家型計畫之最優先計畫,因為微藻含高量的油且不必使用農地也可不使用淡水資源。然現今微藻產油仍存在成本過高之問題。為使得微藻產油能產業化,高油產之藻種是必須的。目前已知環境因子像光強度、日照長短、及多種逆境,皆會影響藻類生長與其油含量及組成;然而這些影響因子的作用機制仍尚待研究,並且由逆境誘發的高油產通常伴隨著對其生長的抑制。為解決此問題以取得高油產之藻株,本子計畫將從生理、分子遺傳、以至基因體學的方法下手。台灣分離擬球藻將為我們的主要目標藻種,因為它具有高油含量之潛能,且為海水藻具環境優勢。為了增加擬球藻類中油脂的含量,擬進行1)光因子(含強度與光週期)對該種藻生長與產油的生理調查,期能增加生物質量與高油脂含量。也將分離不受光強度與光週期影響之快速生長、高油脂的突變體,以有助於大規模的藻類培殖,提高生質能源產量;2)逆境因子(含缺氮與缺磷)對微藻生長與產油的生理調查與建構逆境下之EST基因庫,以期進行系統性快速功能檢測並篩選到對產油調節之關鍵基因。如此將有助於對微藻產油之了解並作為基因改造之用;3)油脂生合成路徑之研究以了解產油之關鍵生合成步驟,並進行澱粉與產油路徑之競爭關係之探討。為加速研究的進行,我們亦將發展新的生技技術及收集基因體資訊於擬球藻上,如篩選合適的篩選標記 (selectable marker) 、引子、以及報導基因。利用分析不同轉殖方法效率高效率的擬球藻轉殖系統亦會被發展。為了進行基因功能性分析及基因改造,RNAi系統亦會被發展。在經費許可的情況下,利用次世代解序方法擬球藻基因體解序亦會被進行。完整的基因體資訊將有助於EST資訊的獲得,並以此更近一步系統性的了解微藻產油生理及調控。我們預期我們的結果將可提供在生質材油生產上具有競爭力的藻種,並提供微藻產油分子及生理機制以為其他子計畫使用而達到實質運用的目標。
Abstract: Biodiesel production from algae is getting more and more attentions because algal culture does not require farm land and freshwater is not required. To achieve the goal for commercial usage, a high oil producing microalgae is a must. It is well known that environmental factors, such as light, nutrient, and stresses, affect the growth of the algae and the accumulation as well as composition of algal lipids. Unfortunately, information on the involved mechanisms and key determinants remains unknown, and stress-enhanced oil production often accompanies with retarded cell propagation. To exploit novel resources for creating algae with high oil content without seriously affecting the growth rate, we aim to understand the key steps for microalgal oil accumulation and genetically manipulate microalgae to achieve their potential oil production. Local marine microalgae, Nannochloropsis oculata, will be our primary target due to its high oil content, with highest at around 70% of dry weight, under stress conditions. We propose to 1) understand the regulation of various light factors, including photoperiod, light intensity, and light quilty, to algal oil accumulation as well as growth rate, and identify mutants that exhibit maximum growth and high oil levels independent of light effects; 2) test stress factors for maximizing oil production in microalgae, systemically identify key factors for oil production, and genetically manipulate microalgae to generate high oil producting algae; 3) determine the key steps in microalgae oil biosythesis pathways, and genetically manipulate these steps to produce algae with high oil content. We will also generate new and required molecular genetic tools in N. oculata to speed up our research progress. In the objective 1), biomass and oil content of N. oculata will be examined under various light conditions. This physiological analysis will enable us to find out the suitable light condition for N. oculata to produce oil. Mutant screening will then be used to identify the N. oculata that accumulated significant amount of oil indepent to the optimal condtion. The muntant will be a valuable asset for biodesiel production. In the objective 2) biomass and oil content of N. oculata will be checked under various stress conditions to find out the best condition for oil accumulation. EST libaries uder stresses and normal culture conditions will be generated to identify key genes for oil accumulation uder the stress condition. The information from ESTs will be used in a systemic screening to find the real key determinates. The results will help us not only to understand the regulation of oil accumulation but also to gain valuable target genes for genetical manipulation. In the objective 3) the key enzymes in triacylglycerol (TAG) biosythesis pathway will directly be examined for their importance in oil accumulation. The balance between starch and lipid biosynthesis is a key for algae to accumulate oil, and therefore, will be examined. To support the above proposed objectives, we will screen for suitable selectable makers, promoters in N. oculata. The transformation system will be optimized by testing various transformation methods, including electroporation, particle bombardment, and agrobacterium-mediated transformation. A gene knockdown will also be set up for functional analysis as well as genetical manipulation. If the funding is sufficient, whole genome sequencing will also be performed using next generation sequencing. We anticipate that our finding and generated high oil production variants will greatly help us setup an ideal culture condition and achieve commercial production of biodiesel from microalgae.