莱茵衣藻光控系统的构建及其在产氢中的应用

发布时间：2018-05-18 04:29

本文选题：莱茵衣藻 + 光合产氢　；参考：《深圳大学》2017年硕士论文

【摘要】：由于氢能是世界上最清洁的能源,它被广泛认为是最理想的化石燃料替代品。因为绿藻的氢酶活性高且具有高效的光合制氢特性,绿藻光合制氢被视为目前最有应用前景的生物制氢途径。然而,由于绿藻氢酶对光合作用放出的氧气非常敏感,使得绿藻持续放氢时间很短,大大制约了绿藻光合制氢产业的发展。目前国际上主要采用更换培养基的“两步法”,通过缺硫培养诱导绿藻持续放氢,但由于藻液分离困难,使二步法无法大规模推广。本实验室通过使用热激诱导调控光合作用的途径,使绿藻可以持续产氢。但热激处理会伤害细胞本身,产生效率也受到影响。本研究以单细胞真核绿藻-莱茵衣藻为材料,建立了一种基于蓝光诱导的莱茵衣藻光控系统并应用于藻细胞产氢调控。该系统由光受体隐花色素CRY2基因及其相互作用蛋白CIB1基因、GAL4转录因子的DNA结合结构域BD和具有转录激活活性单纯疱疹病毒的激活域VP64、UAS上游转录激活序列、特异性靶向PSII复合体功能基因的miRNAs组成。具体研究内容与结果如下:(1)靶向的基因为D1蛋白的编码基因(光系统II核心蛋白),运用WMD3在线平台成功设计出靶向该基因的amiRNA-D1序列;(2)构建了pDb124-GAL4BD-CIB1和pDh124-VP64-CRY2-UAS-ami RNA-D1的表达载体,,通过“珠磨法”对莱茵衣藻进行遗传转化并筛选到表达GAL4BD-CIB1、VP64-CRY2和amiRNA的转基因藻。(3)蓝光诱导处理后,转化株中amiRNA-D1的表达量对比对照组提高了16倍,而野生株CC849表达量相对很低。(4)蓝光诱导后,转化株靶基因D1的表达量下调了78%,靶基因的显著下调表明光控系统启动,amiRNA-D1被表达;野生株CC849的D1基因表达并无明显变化。(5)蓝光诱导处理后,转amiRNA-D1基因藻株较野生株CC849产氢量提高了5倍。由此可见,本研究建立了蓝光诱导调控的光控系统,并基于人工microRNA技术构建了能高效放氢的转基因藻,通过间断蓝光诱导,藻细胞可以实现持续放氢的目标。本研究结果为绿藻生物制氢技术及产业发展提供了新的技术途径。
[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.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ116.2;Q943.2

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