豆血红蛋白基因lba的克隆及其转化衣藻叶绿体

降低细胞内的氧气含量是提高莱茵衣藻产氢效率的重要手段之一。本研究首次尝试将豆血红蛋白基因lba转入衣藻叶绿体中表达,利用豆血红蛋白具有与氧可逆结合的特性,期望降低转基因衣藻细胞内的氧气含量,达到提高衣藻产氢效率的目的。实验结果证明,lba成功转入到衣藻叶绿体中,且对其生长没有产生显著影响,这为下一步调控Lba在衣藻叶绿体中表达活性和提高衣藻产氢效率奠定了实验基础。     

转基因莱茵衣藻hemHc-lbac 和根瘤菌共培养提高产氢培养条件的优化

转基因莱茵衣藻hemHc-lbac(transgenic Chlamydomonas reinhardtii hemHc-lbac)以不同比例与日本慢生大豆根瘤菌(Bradyrhizobium japonicum)混合, 在不同光照条件下进行产氢培养, 以确定产氢的最优条件和探索产氢提高的机理。结果表明藻菌共培养的最优产氢条件为25 ℃、光照30 μE⋅m–2⋅s–1、生长至饱和期的菌和藻体积比为1: 80, 产氢量达到最大, 约为278 μmol⋅mg–1Chl, 是对照组80 μmol⋅mg–1Chl 的3.5 倍。藻菌共培养提高产氢量的主要原因是体系中氧气浓度的降低而使氢化酶活性提高、以及衣藻生物量的增加。该研究为利用藻菌共培养及转基因的方法提高微藻光合生物制氢效率提供了重要实验基础。     

小球藻和莱茵衣藻原生质体的电转化研究

以小球藻及莱茵衣藻原生质体为受体细胞,利用电击法将质粒p CAMBIA1301转入小球藻和莱茵衣藻,摸索电击转化条件并进行分子检测。结果表明:两类藻都对潮霉素敏感,小球藻及莱茵衣藻分别在含25 mg/L和100 mg/L潮霉素的固体培养基上的生长被完全抑制;小球藻和莱茵衣藻原生质体电击转化的最佳电击场强分别为0.8 k V/cm和0.6 k V/cm,最佳脉冲时间均为10 ms;制备原生质体和通过2-脱氧-D-葡萄糖处理可明显提高转化效率;分子检测说明GUS报告基因成功转入两种藻并可稳定遗传。     

Hsp70A-RBCS2融合启动子调控PHB合成酶基因在莱茵衣藻中的表达

利用Hsp70A-RBCS2融合启动子构建了新型的莱茵衣藻表达载体,并获得了聚-β-羟基丁酸（PHB）合成酶基因（phbC）的衣藻表达载体p105C124和pH105C124.通过＂珠磨法＂分别将上述两个衣藻表达载体导入细胞壁缺陷的莱茵衣藻CC-849（Chlamydomonas reinhardtii CC-849）中,得到了具有Zeomycin抗性的转基因藻株.Hsp70A-RBCS2启动子介导的外源基因遗传转化效率明显高于RBCS2启动子,Southern杂交结果显示,phbC基因以低拷贝数整合进莱茵衣藻的基因组DNA中.在光照下,Hsp70A-RBCS2融合启动子能够有效调控phbC基因在莱茵衣藻中的转录和翻译,得到的蛋白产物具有PHB合成酶活性,40℃热激诱导可使PHB合成酶的酶活性提高到1.8倍.因此,Hsp70A-RBCS2融合启动子可使phbC基因在莱茵衣藻中实现可诱导的表达,该研究对利用衣藻合成PHB具有重要的学术意义,进一步的研究将通过＂共转化＂法获得二价或三价的转基因藻,最终实现在转基因藻中合成PHB.     

Cr6+胁迫对莱茵衣藻光合作用的影响

以莱茵衣藻(Chlamydomonas reinhardtii)为研究材料,采用氧电极和快速叶绿素a荧光诱导动力学方法研究了不同浓度和时间Cr6+处理对其光合作用的影响.结果表明:当Cr6+浓度大于40 μmol/L时,莱茵衣藻细胞数逐渐下降,而藻细胞变大;表观光合速率成为负值,呼吸作用随Cr6+处理浓度的增加先上升后下降至对照水平;莱茵衣藻有活性放氧复合体比例随Cr6+处理浓度的增加逐渐降低,80 μmol/L Cr6+处理3 d时已下降至13.72%;光合驱动力(DFABS)随Cr6+浓度增加逐步下降,并以DFφPo在DFABS的下降中的贡献最大.研究发现,重金属Cr6+胁迫显著影响莱茵衣藻的光合作用,而对呼吸作用则影响较小;Cr6+主要通过损伤供体侧的放氧复合体以及阻断QA至QB的电子传递而抑制光系统Ⅱ的功能;莱茵衣藻光系统Ⅱ对Cr6+处理比较敏感且存在着多个作用位点,并首先影响反应中心光能捕获效率,其次影响反应中心的活性,最后影响QA-之后的电子传递.     

RNA干扰20S蛋白酶体α亚基基因对莱茵衣藻油脂代谢的影响

为了解20S蛋白酶体α亚基(20S proteasome alpha subunit A, POA1)基因对莱茵衣藻(Chlamydomonas reinhardtii)油脂代谢的调控,对莱茵衣藻CC425在低氮胁迫下的CrPOA1表达进行了分析,克隆POA1同源基因片段,构建pMaa7IR/XIR干涉载体并转化莱茵衣藻CC425,对CrPOA1进行有效沉默,测定转基因藻株细胞干质量和油脂含量;克隆全长基因,构建CrPOA1-GFP融合表达载体并转化洋葱表皮细胞,进行亚细胞定位。结果表明,莱茵衣藻在低氮培养下,CrPOA1 mRNA水平比对照(正常培养)显著降低(P0.01),RNAi转基因藻株CrPOA1 mRNA水平比对照pMaa7IR/XIR(maa7)降低79.36%～85.35%,沉默效果较好;RNAi转基因藻株细胞干质量与对照maa7无显著差异,油脂含量比对照maa7显著降低6.38%～24.63%,CrPOA1正向调控莱茵衣藻油脂;亚细胞定位于细胞核。这表明CrPOA1参与了莱茵衣藻的油脂代谢过程。     

盐胁迫状态下莱茵衣藻849光合特性的初步研究

以莱茵衣藻（Chlamydomonas reinhardtii）为研究对象,对其在不同NaCl浓度胁迫条件下的光合特性进行了初步研究。结果表明0.04 mol·L^-1的NaCl对莱茵衣藻的生长无显著影响,而0.075 mol·L^-1的NaCl使莱茵衣藻生长速率下降了50%。在低光照强度下,NaCl胁迫下的衣藻光合速率和呼吸速率在最初7 h内比对照组高;而在较高光照强度下,NaCl胁迫下的衣藻的光合速率和呼吸速率均比对照组低;而且光合速率和呼吸速率升高和下降的幅度与NaCl浓度成正比。     

悬浮泥沙与斜生栅藻(Scenedesmus obliquus)对亚洲苦草(Vallisneria natans)光合放氧速率的影响

光照强度是沉水植物生长的主要限制因子。采用Clark 型氧电极研究了悬浮泥沙与斜生栅藻(Scenedesmusobliquus)共同作用对亚洲苦草(Vallisneria natans)光合放氧速率的影响。将亚洲苦草种植于添加不同浓度悬浮泥沙(0.1、0.2、0.5、1.0g·L^–1)和斜生栅藻(105、106、107、108 cell·L^–1)水体中, 定期测定水体中斜生栅藻叶绿素a、光照强度及亚洲苦草光合放氧速率。结果表明: (1)各实验组斜生栅藻呈“S”型增长, 且到达稳定期时间与悬浮泥沙浓度正相关, 与藻细胞添加浓度负相关; (2)除对照组外, 各实验组水体中光照强度曲线均呈先上升后下降变化趋势, 第3 天各实验组的光照强度均出现了高峰值, 而第3 天以后, 光照强度均开始下降, 其中下降最快的是0.1g·L^–1 悬浮泥沙含量实验组, 15 d 后该组平均下降了57.8%, 与空白对照组相比, 差异显著(P<0.05); (3)随着时间推移, 各实验组亚洲苦草光合放氧速率均呈下降的趋势, 其中悬浮泥沙浓度1.0 g·L^–1、藻细胞浓度为108 cell·^L–1 实验组下降最明显, 15 d 后降到了10.88 μmol·^g–1(FW)·h^–1; (4)由可重复双因子方差分析可知悬浮泥沙和斜生栅藻对苦草光合放氧速率的影响均极显著(P<0.01), 且添加不同藻细胞密度造成的组间差异明显大于悬浮泥沙含量造成的组间差异, 这说明亚洲苦草光合放氧速率变化对藻细胞密度变化比悬浮泥沙含量变化更为敏感。研究结果进一步揭示了富营养化泥沙水体苦草群落衰退原因, 且为沉水植被恢复工程提供了科学依据。     

转基因衣藻对重金属的抗性及对镉离子的富集

目的：研究了转基因衣藻对不同重金属离子的抗性和对Cd^2＋的富集行为。方法：将MT-like基因在莱茵衣藻细胞中的表达后通过摇瓶实验测试其对重金属离子的抗性和富集能力。结果：转基因衣藻对Pb^2＋、Zn^2＋和Cd^2＋三种重金属离子的抗性得到明显增强,其中以对Zn^2＋的抗性增强最为显著。转基因藻对Cd^2＋的富集能力经MT-like蛋白表达后较野生藻细胞有较大增加,最大达到144．48μmol／g,为野生藻的8．3倍。结论：转基因衣藻对重金属离子的抗性和对Cd^2＋的富集能力较野生衣藻有较大提高。     

绿色微囊藻的混合营养生长

研究绿色微囊藻(Microcystis viridis)在混合条件下的生长特性,以及葡萄糖,光照强度和pH对绿色微囊藻生长的影响。结果表明:绿色微囊藻混合营养生长与光能自养生长相比,生长速率明显提高,对数期延长,生物量显著提高;随着初始葡萄糖浓度在0~18.0g/l范围内增加,同一光照条件下明显提高了藻细胞的生长速率,但在初始葡萄糖浓度18.0~36.0g/l范围内,同一光照条件下葡萄糖浓度的高低对藻细胞的生长速率不再有更大的影响;在光照强度24~112μE·m^-2·s^-1范围内,初始葡萄糖浓度相同条件下藻细胞的生长速率及对葡萄糖的藻体得率都随光照强度的增强而增大,但当光照强度在112~200μE·m^-2·s^-1时,绿色微囊藻的生长速率增加幅度较小,出现了光饱和现象;当pH处于8.0~10.0间,明显促进了绿色微囊藻的生长,偏离该范围越大,越抑制绿色微囊藻的生长,甚至导致死亡。     

环境因子对小球藻(Chlorella sp. XQ-20044)光合作用的影响

小球藻（Chlorella sp.XQ-20044）是一株具有应用潜力的产油微藻,本文利用测定净光合放氧速率的方法研究了光照强度、温度、pH值和盐度对其光合作用的影响。研究结果表明：小球藻适宜的光照强度为500~1200 μmol·m^-2·s^-1,光补偿点约30 μmol·m^-2·s^-1,光饱和点在600 μmol·m^-2·s^-1附近;光合作用适宜的温度范围为30~42.5℃,最适温度为40℃;适宜的pH值范围7.0~10.0,最适pH值为8.0;适宜盐度范围0.1~0.3 mol/L,最适盐度为0.2 mol/L。从光合作用特性来看,小球藻能适应较强的光照强度、较高的温度、偏碱性和较高的盐度环境,其中可耐受较高盐度的特性,有助于预防敌害生物的污染,对于实现规模培养,特别是利用开放系统进行规模培养较为有利。     

Modeling and optimization of photosynthetic hydrogen gas production by green alga Chlamydomonas reinhardtii in sulfur-deprived circumstance

Biological hydrogen production by the green alga Chlamydomonas reinhardtii under sulfur-deprived conditions has attracted great interest due to the fundamental and practical importance of the process. The photosynthetic hydrogen production rate is dependent on various factors such as strain type, nutrient composition, temperature, pH, and light intensity. In this study, physicochemical factors affecting biological hydrogen production by C. reinhardtii were evaluated with response surface methodology (RSM). First, the maximum specific growth rate of the alga associated with simultaneous changes of ammonium, phosphate, and sulfate concentrations in the culture medium were investigated. The optimum conditions were determined as NH(4+) 8.00 mM, PO(4)(3-) 1.11 mM, and SO(4)(2-) 0.79 mM in Tris-acetate-phosphate (TAP) medium. The maximum specific growth rate with the optimum nutrient concentrations was 0.0373 h(-1). Then, the hydrogen production rate of C. reinhardtii under sulfur-deprivation conditions was investigated by simultaneously changing two nutrient concentrations and pH in the medium. The maximum hydrogen production was 2.152 mL of H(2) for a 10-mL culture of alga with density of 6 x 10(6) cells mL(-1) for 96 h under conditions of NH(4)(+) 9.20 mM, PO(4)(3-) 2.09 mM, and pH 7.00. The obtained hydrogen production rate was approximately 1.55 times higher than that with the typical TAP medium under sulfur deficiency.     

Hydrogen production by the newly isolated Clostridium beijerinckii RZF-1108

A fermentative hydrogen-producing strain, RZF-1108, was isolated from a biohydrogen reactor, and identified as Clostridium beijerinckii on the basis of the 16S rRNA gene analysis and physiobiochemical characteristics. The effects of culture conditions on hydrogen production by C. beijerinckii RZF-1108 were investigated in batch cultures. The hydrogen production and growth of strain RZF-1108 were highly dependent on temperature, initial pH and substrate concentration. Yeast extract was a favorable nitrogen source for hydrogen production and growth of RZF-1108. Hydrogen production corresponded to cell biomass yield in different culture conditions. The maximum hydrogen evolution, yield and production rate of 2209 ml H₂/l medium, 1.97 mol H₂/mol glucose and 104.20 ml H₂/g CDW h⁻¹ were obtained at 9 g/l of glucose, initial pH of 7.0, inoculum volume of 8% and temperature of 35 °C, respectively. These results demonstrate that C. beijerinckii can efficiently produce H₂, and is another model microorganism for biohydrogen investigations.     

Role of Light Intensity and Temperature in the Regulation of Hydrogen Photoproduction by the Marine Cyanobacterium Oscillatoria sp. Strain Miami BG7

The effects of several key environmental factors on the development and control of hydrogen production in the marine blue-green alga (cyanobacterium) Oscillatoria sp. strain Miami BG7 were studied in relation to the potential application of this strain to a bio-solar energy technology. The production of cellular biomass capable of evolving hydrogen gas was strongly affected by light intensity, temperature, and the input of ammonia as a nutrient. Depletion of combined nitrogen from the growth media was a prerequisite for the initiation of hydrogen production. Maximum hydrogen-producing capability coincided with the end of the linear phase of growth. Hydrogen production exhibited considerable flexibility to environmental extremes. The rate of production saturated at low light intensities (i.e., 15 to 30 μEinsteins/m² per s), and no photoinhibition was observed at high light intensity (i.e., 1,000 μEinsteins/m² per s). The upper temperature limit for production was 46°C. Above the light compensation point for O₂ evolution H₂ production was inhibited. However, this problem was alleviated by two related phenomena. (i) The capacity of cells to evolve oxygen deteriorated with increasing culture age and nitrogen depletion, and (ii) the ability of these cells to produce oxygen in closed anaerobic hydrogen production systems was temporally limited.     

Photosynthesis and respiration in a fast growing strain of Gigartina exasperata (Harvey and Bailey)

Photosynthesis and respiration rates of blades from a selected, fast growing strain of the marine red alga. Gigartina exasperata Harvey and Bailey, a carrageenan producer, were measured with an oxygen electrode and compared with rates similarly obtained from wild material of the same species. The measurements, expressed as μl O₂ · mg chl a^?1, min^?1. were made over a light intensity range from 5 to 800 μE · m^?2 · sec^?1 and a temperature range of 6 to 16°C. The photosynthesis light intensity data are best described by hyperbolic functions.     

串联抗菌肽Cecropin B基因在莱茵衣藻中的表达及其抗菌活性分析

为了应对各种抗生素在水产养殖业所带来的副作用,我们在本文中尝试利用微藻对一种抗菌肽进行表达的可行性研究．根据莱茵衣藻核基因组偏爱密码子对抗菌肽Cecropin B基因进行改造,并将4个经改造的Cecropin B基因依次串联起来,中间加上莱茵衣藻的自剪切连接肽序列LWMRFA,人工合成总长度为522 bp的串联Cecropin B基因．将串联Cecropin B基因克隆到含hsp70-RBCS2启动子和RBCS2终止子的pH105载体上,再与携带ble筛选基因的表达框架连接,构建重组表达载体pCB124．采用玻璃珠转化法将载体pCB124导入莱茵衣藻cc-849中,筛选得到能表达抗菌肽Cecropin B的转基因衣藻．经过6个月的保持培养后,进一步对转基因藻细胞提取液进行抗菌活性分析,发现转基因藻具有明显的抑制革兰氏阴性菌(大肠杆菌)和革兰氏阳性菌(枯草芽孢杆菌和溶壁微球菌)生长的特征．这一结果为具有抗菌活性的饵料藻的生产和应用提供了新的途径．     

Separation and Quantitation of 2,3-Butanediol Isomers ((−), (+), and meso) by a Combined Use of Enzyme and Gas Chromatography

Dark hydrogen production by a newly isolated marine green alga, Chlamydomonas MGA 161 was studied. This alga was a halotolerant, not a halophilic type, and grew well in both natural and artificial seawater media. From an experiment with cycloheximide addition, it was found that the hydrogenase reaction in this alga was not a rate-limiting step of dark hydrogen evolution. Starch accumulation increased at a low NH₄Cl concentration (0.5 mm), at a low temperature (20°C), or at a high NaCl concentration (7%). Hydrogen evolution was correlated with starch degradation rather than starch accumulation, and the molar yield of hydrogen from starch-glucose was very high, at about 2 (mol H₂/mol glucose). A comparison of the distribution pattern of fermentation products in various green algae, showed a unique fermentation pattern for Chlamydomonas MGA 161, with high hydrogen evolution but almost no formate.     

Photoproduction of hydrogen by adapted cells of Chlorella pyrenoidosa

Photoproduction of hydrogen gas by the green alga Chlorella pyrenoidosa was studied in a large scale culture of 2.1. Hydrogen was produced by adding sodium hydrosulfite directly to an algal suspension after anaerobiosis in darkness for activation of hydrogenase. The hydrogen production rate showed a characteristic course of an initial burst of gas then steady production, and this course appeared most clearly at cell concentrations around 0.6–0.7 kg/m³. In the final third phase, the hydrogen production rate gradually decreased until evolution ceased. The steady hydrogen evolution was inhibited 75% by a herbicide, DCMU, which blocks electron flow through photosystem II, indicating that the electron donor for hydrogen production was mainly water. The average light intensity within the culture vessel was measured with a diffusing sphere photoprobe. The rate of hydrogen evolution increased hyperbolically with the average light intensity. The duration of hydrogen photoproduction was shorter at higher light intensity due to the inhibition of hydrogenase by concomitantly released oxygen. The duration was shorter also at higher concentrations of algal suspension. It was foudd that the optimum concentration of algae, about 0.7 kg/m³ in this system, must be selected to maximize the yield of hydrogen.     

Survival of human metallothionein-2 transplastomic Chlamydomonas reinhardtii to ultraviolet B exposure

Solar ultraviolet (UV) radiation has a great influence on green organisms,especially planktonlike Chlamydomonas.A human metallothionein-2 gene,which is generally considered to have an anti-radiationfunction by its coding product,was transferred into the chloroplast genome of Chlamydomonas reinhardtii.To dynamically measure the UV effects on Chlamydomonas cells grown in liquid tris-acetate-phosphatemedium,a new method was developed based on the relationship between the chlorophyll content of an algalculture and its absorbance at 570 nm after the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideassay.In this experiment,both the wild-type and the transplastomic C.reinhardtii ceils were cultivated in 96-well microplates containing liquid tris-acetate-phosphate medium in the absence or presence of zinc,copper,cadmium and cysteine.The transgenic C.reinhardtii showed a higher resistance than wild-type to UV-Bexposure under all the examined conditions.Metals in the medium had positive impacts on both types of cells,but had significant influence only on the transplastomic cells.However,the high cell viability of the trans-genie alga at the end of the 8 h UV-B treatment disappeared after a 20-h recovery culture.Cysteine did notprotect cells from UV-B damage,but clearly enhanced the growth of both wild-type and transgenic C.reinhardtii.