本文選題：萊茵衣藻 + 光合產(chǎn)氫　； 參考：《深圳大學(xué)》2017年碩士論文

【摘要】：由于氫能是世界上最清潔的能源,它被廣泛認(rèn)為是最理想的化石燃料替代品。因?yàn)榫G藻的氫酶活性高且具有高效的光合制氫特性,綠藻光合制氫被視為目前最有應(yīng)用前景的生物制氫途徑。然而,由于綠藻氫酶對光合作用放出的氧氣非常敏感,使得綠藻持續(xù)放氫時(shí)間很短,大大制約了綠藻光合制氫產(chǎn)業(yè)的發(fā)展。目前國際上主要采用更換培養(yǎng)基的“兩步法”,通過缺硫培養(yǎng)誘導(dǎo)綠藻持續(xù)放氫,但由于藻液分離困難,使二步法無法大規(guī)模推廣。本實(shí)驗(yàn)室通過使用熱激誘導(dǎo)調(diào)控光合作用的途徑,使綠藻可以持續(xù)產(chǎn)氫。但熱激處理會傷害細(xì)胞本身,產(chǎn)生效率也受到影響。本研究以單細(xì)胞真核綠藻-萊茵衣藻為材料,建立了一種基于藍(lán)光誘導(dǎo)的萊茵衣藻光控系統(tǒng)并應(yīng)用于藻細(xì)胞產(chǎn)氫調(diào)控。該系統(tǒng)由光受體隱花色素CRY2基因及其相互作用蛋白CIB1基因、GAL4轉(zhuǎn)錄因子的DNA結(jié)合結(jié)構(gòu)域BD和具有轉(zhuǎn)錄激活活性單純皰疹病毒的激活域VP64、UAS上游轉(zhuǎn)錄激活序列、特異性靶向PSII復(fù)合體功能基因的miRNAs組成。具體研究內(nèi)容與結(jié)果如下:(1)靶向的基因?yàn)镈1蛋白的編碼基因(光系統(tǒng)II核心蛋白),運(yùn)用WMD3在線平臺成功設(shè)計(jì)出靶向該基因的amiRNA-D1序列;(2)構(gòu)建了pDb124-GAL4BD-CIB1和pDh124-VP64-CRY2-UAS-ami RNA-D1的表達(dá)載體,,通過“珠磨法”對萊茵衣藻進(jìn)行遺傳轉(zhuǎn)化并篩選到表達(dá)GAL4BD-CIB1、VP64-CRY2和amiRNA的轉(zhuǎn)基因藻。(3)藍(lán)光誘導(dǎo)處理后,轉(zhuǎn)化株中amiRNA-D1的表達(dá)量對比對照組提高了16倍,而野生株CC849表達(dá)量相對很低。(4)藍(lán)光誘導(dǎo)后,轉(zhuǎn)化株靶基因D1的表達(dá)量下調(diào)了78%,靶基因的顯著下調(diào)表明光控系統(tǒng)啟動,amiRNA-D1被表達(dá);野生株CC849的D1基因表達(dá)并無明顯變化。(5)藍(lán)光誘導(dǎo)處理后,轉(zhuǎn)amiRNA-D1基因藻株較野生株CC849產(chǎn)氫量提高了5倍。由此可見,本研究建立了藍(lán)光誘導(dǎo)調(diào)控的光控系統(tǒng),并基于人工microRNA技術(shù)構(gòu)建了能高效放氫的轉(zhuǎn)基因藻,通過間斷藍(lán)光誘導(dǎo),藻細(xì)胞可以實(shí)現(xiàn)持續(xù)放氫的目標(biāo)。本研究結(jié)果為綠藻生物制氫技術(shù)及產(chǎn)業(yè)發(fā)展提供了新的技術(shù)途徑。
[Abstract]:Because hydrogen is the cleanest energy in the world, it is widely regarded as the ideal alternative to fossil fuels. Because of its high activity of hydrogen enzyme and high efficiency of photo-hydrogen production, green algae is regarded as the most promising biological hydrogen production pathway. However, due to the sensitivity of green algae hydrogenase to the oxygen released by photosynthesis, the continuous hydrogen release time of green algae is very short, which greatly restricts the development of green algae hydrogen production industry. At present, the "two-step method" of replacing culture medium is mainly used in the world to induce the continuous hydrogen release of Chlorophyta through sulfur-deficiency culture. However, due to the difficulty of separation of algae liquid, the two-step method cannot be popularized on a large scale. In our laboratory, green algae can produce hydrogen continuously by means of heat shock induced regulation of photosynthesis. However, heat shock treatment can damage the cells themselves, and the efficiency of production is also affected. In this study, a photocontrol system of Chlamydomonas rhinoides based on blue light was established and applied to the regulation of hydrogen production by single cell eukaryotic Chlamydomonas rhinorrhoeae (Chlamydomonas rhinorrhoeae). The system was composed of the DNA binding domain BD of the photoreceptor cryptoanthocyanin CRY2 gene and its interacting protein CIB1 gene, GAL4 transcription factor, and the upstream transcription activation sequence of the activation domain VP64UAS of herpes simplex virus with transcriptional activation activity. The miRNAs composition of the functional gene targeting the PSII complex was specifically targeted. The specific research contents and results are as follows: the target gene is D1 protein encoding gene (Photosystem II core protein, using WMD3 online platform to successfully design the amiRNA-D1 sequence targeting this gene, Guan2) to construct the expression vector of pDb124-GAL4BD-CIB1 and pDh124-VP64-CRY2-UAS-ami RNA-D1. After genetic transformation of Chlamydomonas rhinorrhoeae was carried out by beadmill method, the transgenic algae expressing GAL4BD-CIB1, VP64-CRY2 and amiRNA were selected after blue light induction. The expression of amiRNA-D1 in the transformed strain was increased by 16 times compared with that in the control group, but the expression of CC849 in the wild strain was relatively low. After induced by blue light, the expression of target gene D1 in the transformed strain was down-regulated by 78%. The significant down-regulation of the target gene indicated that the light control system initiated the expression of amiRNA-D1. There was no significant change in D1 gene expression of wild strain CC849. After treated with blue light, the hydrogen production of transgenic amiRNA-D1 strain was 5 times higher than that of wild strain CC849. Therefore, the photocontrol system of blue light induced regulation was established, and transgenic algae were constructed based on artificial microRNA technology. Through intermittent blue light induction, algal cells could achieve the goal of continuous hydrogen release. The results of this study provide a new technical approach for biohydrogen production and industrial development of green algae.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TQ116.2;Q943.2

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