集胞藻PCC6803中S2P蛋白酶Slr1821参与热胁迫响应的研究

随着全球CO2排放量不断上升,温室效应日益加剧,全球气候变暖对农作物生长甚至生态环境的破坏日渐严重,研究光合自养生物对热胁迫的响应机制有科学意义和应用前景。集胞藻PCC6803是一种研究光合作用和非生物胁迫响应等生物过程的模式蓝细菌,本文通过同源双交换完全敲除集胞藻PCC6803的S2P蛋白Slr1821的编码基因,构建了缺失突变体Δslr1821,发现经过高温44℃热处理后,突变体完全不能生长,而野生型能够快速恢复热损伤,揭示Slr1821蛋白在热胁迫适应中发挥重要的作用。通过研究藻胆蛋白相对含量和叶绿素含量的变化,发现该基因缺失抑制了细胞热处理后的光合色素包括藻胆蛋白和叶绿素的重新合成,为研究光合自养生物适应热胁迫调控机制提供了新的视角和思路。     

基于代谢组学分析Slr0643蛋白在调控集胞藻PCC6803葡萄糖混养适应中的重要性

本研究文采用集胞藻PCC6803野生型藻株和Slr0643敲除突变体(Δslr0643)藻株,通过生理实验和基于气相色谱-质谱联用(GC-MS)技术的植物代谢组学分析,对Slr0643蛋白调控集胞藻PCC6803葡萄糖混养适应的机制进行探讨.在2.5 mM葡萄糖混养条件下,野生型藻株的生长速率较光自养条件明显加快,而Δ...     

集胞藻6803中S2P同源基因sll0528在多种胁迫下的表达谱分析

为考察集胞藻6803中S2P同源蛋白sll0528的功能,应用实时荧光定量PCR技术研究sll0528基因在各种胁迫条件下不同时间点的表达谱变化。结果表明:sll0528在多种胁迫条件下被显著诱导,根据表达谱特点不同可分为以下三组(1)对高光、双氧水、山梨醇和葡萄糖混养等胁迫的响应属于早期响应,表达量诱导上调的峰值出现在0.5 h内,前三者在0.25 h分别上调约10、80和100倍,后者在0.5 h上调9倍左右;(2)对高温和低温胁迫的响应属于中期响应,前者在1 h上调30倍,后者在2 h显著上调280倍;(3)对酸和盐胁迫的响应属于后期响应,在6 h分别上调90和190倍。由此推断,sll0528可能在响应多种外界环境胁迫中起到重要作用,在不同的胁迫条件下可能参与了不同的信号转导途径从而诱导表达的峰值出现时间和数量有所不同。本研究结果为进一步探讨sll0528基因在胁迫条件下的生理功能及其作用的分子机制奠定了基础。     

裂壶藻突变株S1的细胞生长及其油脂合成的超微结构

本研究以裂壶藻野生株ATCC20888和其突变株S1为研究对象,通过透射电子显微镜观察细胞的生长和胞内油脂产生情况,比较分析了二者的细胞生长及胞内油脂合成规律。结果表明:突变株S1的细胞生长发育及油脂形成速度显著高于野生株,突变株S1藻细胞从生长第1 d起即明显可见单个藻细胞生长,随着培养过程的进行,S1细胞体积基本不变,并且难以看出囊膜包裹的多个藻细胞整体;细胞生长初期,S1胞内油脂呈单个透明状颗粒,随后油滴体积逐渐增大、连结成块、颜色呈深黑色,并可见片状油脂,基本充满整个细胞。本研究从显微水平揭示了不同藻株细胞生长和油脂合成的变化规律,科学解释了不同细胞油脂产量差异的原因,为后续深入理解裂壶藻的细胞生长过程和油脂合成机理提供了一定指导。     

产油微藻的分离筛选与鉴定

微藻所含的油脂是制备生物柴油的理想原料。为了筛选具有产油能力的微藻,作者从自然界水体中分离出8株微藻,对他们的生长特性和产油能力进行评价,旨在筛选出生长速度快、生物量高、油脂含量高的优良藻种。实验结果得到一种油脂产率较高的CC-B3藻株,它的总脂质量分数为36.30%,生物量达到2.43 g/L,油脂产率为62.9 mg/(L·d)。符合优良藻种的条件,是一株高效产油微藻。通过18s r DNA鉴定,该藻株为斜生栅藻(Scenedesmus obliquus),构建了该藻株的进化树。     

产油微藻的筛选及油脂含量测定

为了促进微藻领域研究和微藻生物燃料的生产,从潍坊地区多种生境中分离、筛选产油微藻,并对产油微藻生长情况和油脂积累情况进行研究。结果表明:共分离出49株微藻,其中产油微藻19株;产油微藻的生物量为1.39~6.36 g/L,油脂含量为11.16%~45.62%;6株油脂产率大于90mg/(L·d)微藻在培养的12~15 d产油能力最高,6株微藻最佳收获时间在培养后的14 d。     

产油脂微藻的分离、鉴定及筛选

微藻油脂是生产生物柴油的重要原料,同时也是生产不饱和脂肪酸的原料来源之一。本文以广州市华南理工大学校内东湖为采样点,筛选鉴定湖中存在的微藻并研究其生长和油脂积累特性,旨在筛选出水体中可能存在的富油微藻以便后续研究分析。研究共筛选出6株绿藻,18Sr DNA鉴定显示6株绿藻中DH1、DH2、DH6均属于小球藻属,DH3、DH4、DH5均属于栅藻属,后分析构建了6株微藻进化发育树。通过藻细胞胞内油脂含量测定,表明DH1、DH2与DH6均能较好地积累油脂,尤其是DH2与DH6两株微藻。培养25 d发现其最大生物量(细胞数)分别达到2.14×10~7个/m L和2.74×10~7个/m L,油脂产量分别达到103.89 mg/L和131.69mg/L,藻细胞油脂百分含量均超过30%,表明该两株微藻是潜在的较为理想的产油微藻,可作为生物柴油制备的备用藻种资源。     

铜对葡萄酒酿酒酵母生长活性的影响

以模拟葡萄汁为培养基,添加CuSO4设置0、0.05 mM、0.10 mM、0.20 mM、0.50 mM和1.00 mM 6个不同的Cu2+浓度,研究铜胁迫对葡萄酒酿酒酵母生长活性的影响.结果表明,铜胁迫能够延缓酿酒酵母的生长,降低酿酒酵母的存活率,导致细胞呼吸缺陷型形成频率的增加.0.05 mM Cu2+对酿酒酵母的生长活性基本没有影响.但是1.00 mM Cu2+对3株酿酒酵母的生长活性均产生了显著影响.通过试验比较发现,铜对酵母生长活性的影响可能与菌株对铜离子的吸附能力有关.     

极大螺旋藻高光合速率藻种的紫外诱变筛选

目的:筛选高光合速率极大螺旋藻藻种。方法:结合自然分离筛选、超声处理、紫外诱变和高CO2条件胁迫等方法进行诱变筛选。结果:得到3株极大螺旋藻突变株KYZ1、KYZ2、KYZ3,通过藻丝形态、生长量、CO2利用率、蛋白质含量、叶绿素含量等因素对比,并采用综合评分法,筛选出1株优质藻株KYZ2。结论:与出发藻株相比具有藻丝长度均明显变长、藻丝个体大、生长速度快、光合速率高、蛋白质和叶绿素含量高等优点,是一株具有工业养殖潜力的藻株。     

氨氮质量浓度和温度对雨生红球藻生长特性的影响

研究了不同氨氮(氯化铵)浓度(0.05~0.8 g/L)和生长温度(22、25、28℃)对雨生红球藻生长特性的影响,以及不同胁迫温度(30、35、38℃)对虾青素积累的影响。测定了不同培养条件下雨生红球藻的OD值、干重、叶绿素以及虾青素含量。研究结果表明:以氯化铵为氮源的条件下,雨生红球藻更适宜在低氨氮浓度下生长,适宜的氨氮质量浓度是0.1 g/L,最适生长温度为22℃,以高温胁迫时,最佳胁迫温度为35℃。     

D-Amino acid dipeptide production utilizing D-alanine-D-alanine ligases with novel substrate specificity

D-Alanine-D-alanine ligase (Ddl) is an important enzyme in the synthesis of bacterial peptidoglycan. The genes encoding Ddls from Escherichia coli K12 (EcDdlB), Oceanobacillus iheyensis JCM 11309 (OiDdl), Synechocystis sp. PCC 6803 (SsDdl) and Thermotoga maritima ATCC 43589 (TmDdl), the genomic DNA sequences of which have been determined, were cloned and the substrate specificities of these recombinant Ddls were investigated. Although OiDdl had a high substrate specificity for D-alanine; EcDdlB, SsDdl and TmDdl showed broad substrate specificities for D-serine, D-threonine, D-cysteine and glycine, in addition to D-alanine. Four D-amino acid dipeptides were produced using EcDdlB, and D-amino acid homo-dipeptides were successfully produced at high yields except for D-threonyl-D-threonine.     

Effects of glucose addition and light on current outputs in photosynthetic electrochemical cells using Synechocystis sp. PCC6714

The effects of glucose addition and light on the current outputs in electrochemical cells using a cyanobacterium Synechocystis sp. PCC6714 were investigated under photo- and chemoheterotrophical conditions. The addition of glucose to the anode solutions of the electrochemical cells resulted in a rapid increase in the current outputs under both light and dark conditions. Although the coulombic outputs were almost the same between under light and dark conditions, the rate of glucose consumption was faster under illumination than in the dark. The total sugar content in the cells of strain PCC6714 increased with the addition of glucose and the total sugar accumulated remained intact during the discharge under illumination, while it decreased gradually in the dark. When the light was switched off after the addition of glucose, the current output markedly increased. The coulombic outputs obtained after darkening were 10 to 80 times larger than that obtained by the addition of glucose under the continuous light or dark conditions. Synechocystis sp. completely incorporated 0.14 mM and 0.42 mM glucose for 1 h and 3 h, respectively, under illumination. There was no difference in the coulombic outputs between 1 h and 12 h illumination times in the electrochemical cells with 0.14 mM glucose. When the light was switched off after 1 h illumination in the electrochemical cells with 0.42 mM glucose, the coulombic output obtained from the electrochemical cell was lower than that in the electrochemical cell with 12 h illumination. This indicates that the current output was produced with higher efficiency with glucose incorporated under illumination than that in the case of glucose incorporated after darkening. The highest coulombic yield of 54% in this experiment was obtained by darkening in the electrochemical cell with 0.14 mM glucose.     

Impact of Sod on the Expression of Stress‐Related Genes in Listeria monocytogenes 4b G with/without Paraquat Treatment

Listeria monocytogenes is a foodborne pathogen that causes listeriosis. Paraquat can generate reactive oxygen species (ROS) in cells, which results in oxidative stress. It was first shown that 1 mM of paraquat inhibited the growth rate of a superoxide dismutase (sod)‐deletion mutant (?sod) generated from L. monocytogenes 4b G but not in the wild‐type, and induced the expression of other resistance genes (kat, fri, perR, sigB, and recA) as well as sod in the wild type. Interestingly, without paraquat treatment the expression of all the 5 genes were repressed in ?sod compared to the wild type, while the expression of recA triggering SOS response, a global response to DNA damage, was increased in ?sod in the presence of 1 mM paraquat. Taken together, these results suggest that SOD plays a central role in oxidant defense of L. monocytogenes 4b G, and SOS probably significantly impacts ?sod survival under oxidative stress.     

PRODUCTION of FLAVOR BYAMUTANT of YEAST

ABSTRACT. Strain CCU-N₁₆-18143 was derived with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment three times from a wild strain of yeast CCU-16 as a mutant to produce more delicious and intense flavors for nutrient beverage from glucose medium. This mutant was identified by computer system as Saccharomyces sp. the optimal culture medium for the production of flavor is one liter of medium containing 100 g glucose, 12 g ammonium nitrate, 3 g yeast extract, 1 g magnesium sulfate, 1 g ammonium sulfate, 2 g potassium dihydrogen phosphate, pH: 3.5. the optimal culture conditions are: temperature: 30C; agitation: 150 rpm., 50 ml medium in 500-ml Hinton flask; incubation time: 96 h. the volatile flavor compounds in the culture medium were analyzed by capillary gas chromatography and capillary gas chromatography-mass spectrometry. It was found that more volatile compounds were produced by mutant strain than wild strain. the content of isoamyl alcohol increased from 7.76 to 61.64%, whereas ethyl alcohol decreased from 85.25 to 5.77%.     

DNA taken into Bacillus subtilis competent cells by lysed-protoplast transformation is not ssDNA but dsDNA

Competent Bacillus subtilis incorporates whole-genome DNA (4215 kb) from the protoplast lysate of B. subtilis subtilis [Akamatsu, T. and Taguchi, H., Biosci. Biotechnol. Biochem., 65, 823-829 (2001)]. A continuous incorporated DNA is longer than 1500 kb [J. Biosci. Bioeng., 101, 257-262 (2006)]. Whether the incorporated DNA is single-stranded (ssDNA) or double-stranded DNA (dsDNA) has been studied by examining the transforming activity of the incorporated DNA. B. subtilis BEST7027 was used as the donor strain, which has a heterologous region consisting of the 145 kb region of the Synechocystis sp. PCC6803 genome and erm gene. The donor DNA was transferred to a wild-type or a recA recipient strain (AYG2 or SYN9), and protoplast lysate was prepared from the transformants and used as the donor DNA source for the second recipient strain (AU1 or AV1). The intergenote region showed a significant transforming activity. When DNase I was added to both cells collected from the first transformation mixture and the following protoplastization, the result was similar to that obtained without DNase I. All of the observations strongly suggest that the incorporated DNA is dsDNA, and the transformation of competent B. subtilis by DNA in protoplast lysate is different from that by purified DNA taken up conventionally.     

烟草DGAT3基因响应冷胁迫应答的功能研究

二脂酰甘油酰基转移酶（Diacylglycerol acyltransferase,DGAT）是催化三脂酰甘油（TAG）合成途径的关键酶,在植物生长发育和逆境胁迫响应中具有重要的作用。本研究从普通烟草品种K326中克隆得到NtDGAT3基因,利用荧光定量PCR技术分析不同组织中和低温胁迫下NtDGAT3的表达规律。同时,利用CRISPR/Cas9技术制作NtDGAT3敲除植株,对其耐冷性进行了分析。结果表明,NtDGAT3在烟草根、茎、叶中均有表达,且叶部表达量最高;NtDGAT3受冷胁迫强烈诱导,说明该基因可能对冷胁迫有响应。低温处理试验结果表明,敲除突变体ntdgat3对冷敏感,突变体叶片离子渗漏率及丙二醛含量的变化幅度显著高于野生型烟草;在ntdgat3中,膜脂代谢关键基因PLDα1的表达水平显著高于野生型烟草,而PLDδ的表达水平显著低于野生型烟草。以上结果表明,NtDGAT3基因通过PLDα1和PLDδ的表达参与调节植物低温响应。     

粗毛革孔菌及其高产漆酶突变株的比较分析

漆酶是一种广泛分布的多酚氧化酶,在食品、能源和环保等领域具有重要的应用价值。本实验室所筛的高产漆酶粗毛革孔菌经诱变后,生长形态变化大,突变株漆酶活力可达303 U/mL,是野生菌的3倍多。本文比较分析了原始菌株和突变菌株的生理生化差异,并通过RAPD比较分析了它们的差异基因。用140条随机引物扩增获得20条稳定的特异条带,并对其中的3条进行回收、克隆,测序结果与BLASTX、NCBI数据库的比对表明引物S381扩增出的809 bp的特异条带对应于云芝的假想蛋白,引物S225扩增出的654 bp的特异条带对应于30 S核糖体蛋白S4,引物S193扩增的1027 bp的特异条带对应于糖苷水解酶。该试验为探讨粗毛革孔菌突变株高产漆酶的分子机制,以及通过定向突变构建高产漆酶工程菌提供理论依据。