杜氏盐藻异养表达载体的构建及异养转化株的鉴定

分别构建杜氏盐藻诱导型和组成型异养表达载体,筛选并初步鉴定异养转化藻株。通过RT-PCR从人胎盘组织中克隆并鉴定人红细胞葡萄糖转运基因(Glut1),构建以诱导型双拷贝碳酸酐酶启动子(DCA)驱动Glut1表达的中间载体,然后与筛选标记Bar盒连接形成盐藻诱导型异养表达载体pMDDGN-Bar。此外,将pU?GUS(简称G5)质粒的GUS基因去除,回收大片段载体后与Glut1基因连接,构建以组成型启动子ubiquitin驱动的组成型异养表达载体G5Glut1-Bar。通过电击转化法转化盐藻,使Glut1得到表达,筛选具有草丁膦(PPT)抗性的表达Glut1的盐藻转化株。提取转化株总RNA,RT-PCR检测目的基因的整合。克隆获得了1479bp的Glut1序列,编码493个氨基酸。电泳检测各酶切结果表明Glut1、DCA、Nos和Bar盒已依次连接到相应的载体上,说明异养表达载体构建成功。经PPT筛选数周后,转化藻株生长良好,而对照野生藻株全部死亡。电泳检测RT-PCR产物表明两株转化株在相应位置(约250bp处)出现了较为特异的扩增条带,Blast同源性分析显示序列与人Glut1基因的同源性为100%。诱导型...     

杜氏盐藻核基质结合区结合蛋白的细胞定位

为研究核基质结合区结合蛋白的功能及调控机制,PCR扩增杜氏盐藻MBP的cDNA全长序列及N端和C端序列,与绿色荧光蛋白基因融合构建真核表达载体,脂质体转染CHO细胞,Western blotting和荧光显微镜检测基因表达情况和细胞定位。结果显示:MBP及N端和C端融合蛋白成功在CHO细胞表达,MBP和C端部分定位于细胞核且聚集于核仁,N端部分分布整个细胞,说明MBP定位于细胞核且细胞定位信号位于C端,MBP可能与rRNA前体结合发挥作用。     

Microalgae as a raw material for biofuels production

Biofuels demand is unquestionable in order to reduce gaseous emissions (fossil CO₂, nitrogen and sulfur oxides) and their purported greenhouse, climatic changes and global warming effects, to face the frequent oil supply crises, as a way to help non-fossil fuel producer countries to reduce energy dependence, contributing to security of supply, promoting environmental sustainability and meeting the EU target of at least of 10% biofuels in the transport sector by 2020. Biodiesel is usually produced from oleaginous crops, such as rapeseed, soybean, sunflower and palm. However, the use of microalgae can be a suitable alternative feedstock for next generation biofuels because certain species contain high amounts of oil, which could be extracted, processed and refined into transportation fuels, using currently available technology; they have fast growth rate, permit the use of non-arable land and non-potable water, use far less water and do not displace food crops cultures; their production is not seasonal and they can be harvested daily. The screening of microalgae (Chlorella vulgaris, Spirulina maxima, Nannochloropsis sp., Neochloris oleabundans, Scenedesmus obliquus and Dunaliella tertiolecta) was done in order to choose the best one(s), in terms of quantity and quality as oil source for biofuel production. Neochloris oleabundans (fresh water microalga) and Nannochloropsis sp. (marine microalga) proved to be suitable as raw materials for biofuel production, due to their high oil content (29.0 and 28.7%, respectively). Both microalgae, when grown under nitrogen shortage, show a great increase (~50%) in oil quantity. If the purpose is to produce biodiesel only from one species, Scenedesmus obliquus presents the most adequate fatty acid profile, namely in terms of linolenic and other polyunsaturated fatty acids. However, the microalgae Neochloris oleabundans, Nannochloropsis sp. and Dunaliella tertiolecta can also be used if associated with other microalgal oils and/or vegetable oils.     

杜氏盐藻寡糖基转移酶亚基STT3a功能结构域的克隆与表达分析

为了研究STT3a基因在杜氏盐藻耐盐及鞭毛再生方面的作用,根据衣藻、拟南芥等STT3a蛋白的氨基酸高度保守序列VCVFTA、DVDYVL设计一对简并引物,采用RT-PCR及3'RACE的方法扩增杜氏盐藻STT3a蛋白功能结构域的cDNA序列。序列分析显示克隆的cDNA全长1650bp,具有一定保守性,与衣藻、拟南芥和人的相似性分别为48%、50%和46%。实时荧光定量PCR结果显示杜氏盐藻STT3amRNA水平随着盐浓度的升高而逐渐增加,其水平在3.5mol/LNaCl浓度时比在1.5mol/LNaCl浓度时升高了11倍(P0.01)。另外,与没有脱鞭毛的杜氏盐藻相比,STT3amRNA在鞭毛再生过程中持续高表达。本研究显示杜氏盐藻STT3a基因的高表达可以增强其盐适应和鞭毛再生能力。     

Mechanism of extraction of beta-carotene from microalga Dunaliellea salina in two-phase bioreactors

We show that it is possible to extract beta-carotene selectively from Dunaliella salina in two-phase bioreactors. The cells continue to produce beta-carotene and the extracted part is substituted by newly produced molecules. This process is called "milking." We performed several experiments to understand the exact mechanism of the extraction process. The results show that direct contact between the cells and the biocompatible organic solvent was not a requirement for the extraction but it accelerated the extraction. Electron microscopy photographs showed an undulated shape of the cell membrane and a space between the cell and the chloroplast membranes in the cells growing in the presence of dodecane (a biocompatible solvent). Extra-chloroplast beta-carotene globules located in the space between the cell and the chloroplast membranes were observed in these cells as well. It was shown that dodecane was taken up by the cells. The concentration of dodecane in the cells was about 13 pg.cll(-1). It can be concluded that dodecane uptake by the cells is responsible for the morphological changes in the cells and leads to more activity in the cell membrane. The results suggest two possible modes of extraction. One of the mechanisms is transport of the globules from the chloroplast to the space between the cell and the chloroplast membranes and subsequently from there to the outside by exocytosis. Another possible mode for the extraction could be release of beta-carotene from the globules as a result of alterations in the membrane in response to the uptake of dodecane. beta-Carotene molecules diffuse from the chloroplast to the space between the cell and the chloroplast membranes and from there to the medium either by diffusion or by exocytosis after accumulation in the vesicles.     

A mutant of the green alga Dunaliella salina constitutively accumulates zeaxanthin under all growth conditions

A novel mutant (zea1) of the halotolerant unicellular green alga Dunaliella salina is impaired in the zeaxanthin epoxidation reaction, thereby lacking a number of the beta-branch xanthophylls. HPLC analysis revealed that the zea1 mutant lacks neoxanthin (N), violaxanthin (V) and antheraxanthin (A) but constitutively accumulates zeaxanthin (Z). Under low-light physiological growth conditions, the zea1 (6 mg Z per g dry weight or 8 x 10(-16) mol Z/cell) had a substantially higher Z content than the wild type (0.2 mg Z per g dry weight or 0.5 x 10(-16) mol Z/cell). Lack of N, V, and A did not affect photosynthesis or growth of the zea1 strain. Biochemical analyses suggested that Z constitutively and quantitatively substitutes for N, V, and A in the zea1 strain. This mutant is discussed in terms of its commercial value and potential utilization by the algal biotechnology industry for the production of zeaxanthin, a high-value bioproduct.     

Stable expression of hepatitis B surface antigen gene in Dunaliella salina (Chlorophyta)

A stable transformation system for theexpression of foreign genes in theunicellular marine green alga Dunaliella salina was established. Amongfive antibiotics, 60 g mL^-1chloramphenicol completely inhibitedgrowth. Of five promoters tested, theubiquitin-$Ohgr; promoter yielded thehighest -glucuronidase (GUS) activity.The hepatitis B surface antigen (HBsAg)gene was introduced into the cells by usingelectroporation. PCR and Southern blotanalysis amd it was shown that the gene wasintegrated into the genome. The stableexpression of HBsAg protein was confirmedby enzyme-linked immunosorbent assay(ELISA) and Western blot analysis. Theintroduced DNA and HBsAg expression weremaintained stable for at least 60generations in medium devoid ofchloramphenicol. This is an important steptoward the production of useful foreignproteins in the alga.     

Molecular Cloning and Stress-Dependent Expression of a Gene Encoding ω3-Fatty Acid Desaturase in the Microalga Dunaliella salina

A fragment of the gene des3-1 encoding 3 fatty acid desaturase was cloned from a cDNA library of the unicellular green galophilic alga Dunaliella salina. The comparative phylogenetic analysis of 3-desaturase amino acid sequences from diverse organisms placed the desaturase of D. salina between cyanobacteria and higher plants in the evolutionary range of desaturases. The expression of des3-1 was studied in D. salina cells exposed to low temperatures, high irradiance, and high CO₂ concentrations. Lowering the external temperature from 32 to 22°C produced a transient increase in the level of specific mRNA. Considerable accumulation of mRNA for 3-desaturase was also observed when CO₂ concentration in gas–air mixture was raised from 2 to 10%. An irradiation increase from 70 to 500 mol/(m² s) did not affect the level of specific mRNA. The latter evidence presumes that in Dunaliella cells, this desaturase is probably located in the endoplasmic reticulum, rather than in the chloroplast.     

Photosystem-II repair and chloroplast recovery from irradiance stress: relationship between chronic photoinhibition,light-harvesting chlorophyll antenna size and photosynthetic productivity in Dunaliella salina (green algae)

High-light (HL) grown Dunaliella salina cells exhibit lower pigment content, a highly truncated chlorophyll (Chl) antenna size, and accumulation of photodamaged PS II centers in the chloroplast thylakoids (chronic photoinhibition). In HL-grown cells, the rate of photosynthesis saturated at higher irradiances and the quantum yield was lower compared to that of normally-pigmented low-light (LL) grown cells. In spite of these deficiencies, the light-saturated rate of photosynthesis for the HL-cells, when measured on a per chlorophyll basis, was 3 times greater than that of the LL-grown cells. To delineate the effect of photoinhibition from the Chl antenna size on quantum yield and rate of photosynthesis, HL-acclimated cells were switched to LL-conditions. Repair of photodamaged PS II, estimated from the recovery of functional PS II centers and from the increase in the quantum yield of photosynthesis, occurred with a half-time of 1 h. Chlorophyll accumulation in the cells occurred with a half-time of 4 h. The differential kinetics in repair versus Chl accumulation provided a window of opportunity, within about 2–3 h after the HLLL shift, when cells exhibited a high quantum yield of photosynthesis, a small Chl antenna size and a light-saturated rate that was 6–9 times greater than that of the normally pigmented LL-grown cells. The work provides insight on the temporal sequence of events at the chloroplast and thylakoid membrane levels, leading from a chronic photoinhibition of PS II to repair and recovery. It is suggested that it is possible to maximize photosynthetic productivity and light utilization in mass microalgal cultures by minimizing the light-harvesting Chl antenna size of the photosystems.