硫酸盐还原菌耐药性研究

硫酸盐还原菌（简称SRB）对杀菌剂十二烷基二甲基等基氯化铵（简称“1227”）的耐药性是由药物诱导产生,这种由适应所产生的耐药性获得快而且是可逆的,随着时间的推迟抗药性减弱,6个月后抗药性逐渐消失。研究还表明SRB对甲硝叹没有明显的交叉耐药性。     

ADAPTIVE FORMATION OF NITRATE REDUCING SYSTEM IN ANABAENA CYLINDRICA

1. Changes in capacities for reducing nitrate, nitrite and hydroxylaminecaused by provision or depletion of various nitrogen sourceswere investigated with a nitrogen fixing blue-green alga, Anabaenacylindrica, and adaptive nature of these reducing system wasdemonstrated.
2. It was found that, under light-aerobic conditions,nitrate-and nitrite- reducing systems were induced by nitrateor nitritebut not by N₂ ammonia and glutamate. On the otherhand, theactivity of enzymes pertaining to hydroxylamine reductionwasstimulated equally by nitrate, nitrite and N₂. The latteractivitywas suppressed markedly in the presence of ammoniaor glutamate.
3. Adaptive formation of nitrite reducing systemis completelyinhibited by chloramphenicol, a potent inhibitorof proteinsynthesis. No formation was also observed under theanaerobiccondition or in the dark.
4. On the basis of thesefindings, a tentative scheme for pathwaysof nitrate reductionand nitrogen fixation in Analaena cylindricawas proposed.

(Received August 22, 1962; )     

小球藻细胞壁缺陷型突变体的筛选及转化系统的建立

由于高效、稳定遗传转化系统的缺乏,小球藻遗传改良以及油脂代谢机理的研究等工作难以进行。研究旨在通过筛选获得小球藻Chlorella vulgaris细胞壁缺陷型突变体,在此基础上建立其转化系统。首先通过紫外诱变获得小球藻Chlorella vulgaris的突变体库,基于细胞壁缺陷型突变体在1% Triton X-100处理后叶绿素会释放到上清中的原理,利用酶标仪高通量地从约4000个突变体中筛选获得10株细胞壁缺陷的小球藻。同时以小球藻内源性-tubulin的启动子和终止子作为启动子和终止子,以AphⅧ (Aminoglycoside 3'-Phosphotransferase type Ⅷ)作为报告基因构建了转化载体pHK203。通过优化电转缓冲液组分和电击参数,确定了细胞壁缺陷型突变体CWD-3的最佳转化条件,即2 g pHK203,ddH2O作为电转缓冲液,1500 V,525 ,50 F的电击条件下,转化效率可达到40个转化子/g DNA。研究为小球藻Chlorella vulgaris的油脂代谢通路和遗传改良提供了技术基础,同时由于可降低破壁成本,筛选获得的细胞壁缺陷型突变体适于工业化生产小球藻藻粉。     

大规模的酵母双杂交系统在蛋白质相互作用组图谱中的应用

蛋白质-蛋白质之间的相互作用是蛋白质发挥其功能的重要途径之一。通过研究蛋白质组中所有蛋白质之间的相互作用做出蛋白质相互作用对图谱是功能基因组时代许多科学家关注的问题,而大规模的酵母双杂交系统是蛋白质相互作用对图谱的研究中应用较为广泛的策略。近两年来该策略最具代表的实例是用它进行酵母中所有蛋白之间相互作用的检查。但是巨大的蛋白质网络比我们想象要大得多,单一的双杂交系统不能解决所有问题,需要同其它的方法有效地结合。     

铜绿微囊藻生物钟蛋白KaiA的自身相互作用研究

正蓝藻虽为原核生物,但它也和真核生物一样具有生物钟,它的固氮作用、光合作用、氨基酸吸收、细胞分裂以及基因表达等生理代谢过程都受到生物钟的调控,具有昼夜节律性。虽然蓝藻生物钟和真核生物钟一样,都以近24h的周期运行,都具有温度补偿效应,光、温等环境因素都能重置生物钟的时相,但组成蓝藻生物钟的钟蛋白与真核生物钟蛋白间不具有任何同源性,蓝藻生物钟的计时机制也与真核生物钟存在差异1-2。蓝藻钟基因为一个基因簇kai,由三个基因kaiA、kaiB、kaiC以单一拷贝成簇排列,Kai蛋白组成蓝藻生物钟的核心即中央振荡器,其中kaiC蛋白的磷酸化状态是中央振荡器产生周期性震荡的关键,它决定中央振荡器的时相,而kaiC的磷酸化状态则受到kaiA和kaiB的调节。kaiA是接受和整合环境信息的钟蛋白,具有N-端和C-端两个结构域,N-端缺乏保守天冬氨酰残基的伪接受域能通过与输入途径的某种蛋白(目前未知)发生相互作用而感受环境信号       

酵母菌杀菌系统的杀菌活性

探讨酵母菌杀菌系统（即３０株杀菌酵母菌）对３８株真菌或细菌的杀菌活性。结果显示杀菌酵母菌不仅对念珠菌、新型隐球菌和烟曲霉菌等真菌有杀菌活性,而且对其不相关的葡萄球菌、肠杆菌科、绿脓杆菌、不动杆菌和副溶血性弧菌也有杀菌活性,提示该系统具有广泛的杀菌活性,特别是Ｋ３６。     

IN SITU SEPARATION OF ETHANOL WITH AQUEOUS TWO-PHASE SYSTEM AND ASSESSMENT OF KLa FOR YEAST GROWTH IN BATCH CULTIVATION

In the fermentation process, the separation of product and its purification is the most difficult and exigent task in the ground of biochemical engineering. Another major problem that is encountered in the fermentation is product inhibition, which leads to low conversion and low productivities. Extractive fermentation is a technique that helps in the in situ removal of product and better performance of the fermentation. An aqueous two-phase system was employed for in situ ethanol separation since the technique was biofriendly to the Saccharomyces cerevisiae and the ethanol produced. The two-phase system was obtained with polyethylene glycol 4000 (PEG 4000) and ammonium sulfate in water above critical concentrations, with the desire that the ethanol moves to the top phase while cells rest at the bottom. The overall mass transfer coefficient (K_La) was also estimated for the yeast growth at different rpm. The concentration and yield of ethanol were determined for conventional fermentation to be around 81.3% and for extractive fermentation around 87.5% at the end of the fermentation. Based on observation of both processes, extractive fermentation was found to be the best.     

FORMATION OF AUXIN IN YEAST CULTURES

We have found far more auxin in the culture media of bakers'' yeast than was obtained by Kögl and Kostermans from the cells themselves. The production of auxin by yeast cells resembles the formation observed in other organisms such as Rhizopus and Rhizobium which also form auxins in their culture media. The auxin yield was found to increase with the concentration of sucrose and to decrease with the concentration of peptone. An inverse relation with the rate of cell multiplication was observed. Enlarged and elongated cells appeared only in those media which contained considerable amounts of auxin. The total auxin yield in the various cultures was found to be directly proportional, below pH 5, to the hydrogen ion concentration. Thus, it was proposed that certain growth conditions favor the breakage of the link between auxin and its protein carrier (Skoog and Thimann) 1940) and consequently accelerate the rate of excretion of auxin into the growth medium.     

三角褐指藻产油突变株的筛选

能源短缺和原油价格上涨是全球关注的问题之一, 生物柴油作为一种优质的替代液体燃料越来越受到重视。以油料作物、废食用油和动物脂肪为原料生产生物柴油已远远不能满足需求。相比而言, 产油微藻具有光合效率高、生物量大、油含量高、生长速率快、不受季节的限制及不占用耕地等优势15, 被认为是制备生物柴油燃料的更有潜力的原料。由于目前微藻生物柴油生产成本过高, 尚未获得商业化生产。       

高等担子菌双重营养缺陷突变菌株的分离

带有单一营养缺陷的凤尾菇和裂褶菌的单核体菌株经亲和性交配,各自交配产生后代,从中分离出遗传特性稳定,生理特征表型正常的双重缺陷营养害变型菌株,为原生质体融合育种研究提供了可靠的亲本菌株。     

酵母双杂合系统在丙型肝炎病毒NS5A研究中的应用

母双杂合系统是在酵母细胞中研究蛋白－蛋白相互作用的新技术。应用该技术,以丙型肝炎病毒（ＨＣＶ）非结构蛋白５Ａ（ＮＳ５Ａ）为“诱饵”,筛检了人中细胞ＨｅｐＧ２来源的ｃＤＮＡ文库,获得了７个ＮＳ５Ａ结合蛋白的基因克隆。报道了其中两个阳性克隆（＃２、＃１６）的结果,并对“人核小体组装蛋白相关蛋白”（Ｈｕｍａｎ ｎｕｃｌｅｏｓｏｍｅ ａｓｓｅｍｂｌｙ ｐｒｏｔｅｉｎ－ｒｅｌａｔｅｄ ｐｒｏｔｅｉｎ,ｈＮＲ     

ROLE OF SULFUR METABOLISM IN COPPER-RESISTANCE OF YEAST

Cells of a normal yeast strain and of its copper-resistant substrainwere more severely injured by copper treatment under sulfurdeficiency than under nitrogen or carbon deficiency. Even underthese starved conditions, the resistant substrain gave higherviable counts than the parent strain after the copper treatment.Several hours were needed for the sulfur-starved cells to recovertheir copper-resistance when resupplied with sulfur. The observed effect of the amount of sulfur supply on the cellyield was not contradictory to the idea that resistant cellsuse for their resistance mechanism a part of sulfur they absorb.Methionine made cells more sensitive to copper, and this effectwas antagonized by ethionine to some extent. The importance of sulfur metabolism in copper resistance isdiscussed. ¹This work was supported by a Grant in Aid for Fundamental ScientificResearch, Ministry of Education, Japan. ²Present address: Konan University, Kobe, Japan. (Received October 8, 1959; )     

THE COST OF SEXUAL SIGNALING IN YEAST

The handicap principle holds that costly sexual signals can reliably indicate mate quality. Only individuals of high quality can afford a strong signal—the cost of signaling is relatively lower for high‐quality signalers than for low‐quality signalers. This critical property is difficult to test experimentally because the benefit of signaling on mating success, and cost of signaling on other components of fitness, cannot easily be separated in obligate sexual organisms. We therefore studied the facultatively sexual yeast Saccharomyces cerevisiae, which produces pheromones to attract potential mates. To precisely measure the cost of signaling, the signal was reduced or removed by deleting one or both copies of the pheromone‐encoding genes and measuring asexual growth rate in competition with a wild‐type signaler. We manipulated signaler quality either by changing the quality of the assay environment or by changing the number of deleterious mutations carried. For both types of treatment, we found that the cost of signaling decreased as the quality of the signaler increased, demonstrating that the yeast pheromone signal has the key property required for selection under the handicap principle. We found that cells of high genetic quality produce stronger signals than low‐quality cells, verifying that the signal is indeed honest.     

鲤春病毒血症病毒糖蛋白酵母表面展示系统的建立

糖蛋白(Glycoprotein, G)作为鲤春病毒血症病毒(Spring Virernia of Carp Virus, SVCV)主要的抗原蛋白, 已成为现阶段SVCV病毒检测、抗体制备以及疫苗研制的热点。为了对其进行酵母表面展示, 研究以SVCV-shlj1分离株基因组为模板, 通过RT-PCR技术, 体外扩增获得SVCV表面糖蛋白的基因开放阅读框(1530 bp)片段, 将其克隆至酵母表面展示载体pYD1, 构建重组质粒pYD1-G。利用电转化方法将重组质粒pYD1-G导入酿酒酵母EBY100感受态细胞, 经YNB选择培养基筛选和菌液PCR的鉴定, 挑选出阳性转化子(命名为EBY100-pYD1-G), 对其进行2%半乳糖诱导。利用细胞免疫荧光和流式细胞仪检测G蛋白的酵母表面展示情况。细胞免疫荧光结果显示, 诱导后的酵母细胞EBY100-pYD1-G能产生特异性红色荧光, 且随着诱导时间的增加, 红色荧光的酵母细胞所占比例不断增加, 各组之间差异显著(P< 0.05)。流式细胞仪检测结果显示, 酵母细胞的荧光强度与诱导时间呈正比, 其中诱导48h与72h的酵母细胞荧光强度不存在显著差异, 基本趋于稳定不变的状态。因此, 选取诱导48h为酵母表面展示的最佳诱导时间。上述研究结果表明SVCV的G蛋白已经成功展示于酿酒酵母细胞表面, 研究为鲤春病毒血症酵母口服疫苗的研发奠定了前期基础。     

海藻糖微生物酶法合成机制的研究

来源于嗜酸热古菌芝田硫化叶菌(Sulfolobus shibatae)B12的麦芽寡糖基海藻糖合酶(MTSase)和麦芽寡糖基海藻糖海藻糖水解酶(MTHase)基因在大肠杆菌中获得表达。将获得纯化的两个酶,分别以麦芽寡糖和淀粉为转化底物,在pH5.5,60℃条件下合成海藻糖。从反应产物分析结果可知,两个酶合成海藻糖时能利用的最小底物是麦芽四糖,海藻糖产率与麦芽寡糖链长正相关。同时还发现两个酶都具有轻微的α-1,4-葡萄糖苷酶活性,能在麦芽寡糖还原末端水解α-1,4糖苷键,生成葡萄糖分子,其反应最小底物分别是麦芽三糖和四糖。推测海藻糖合成酶可能有两个不同的催化活性中心。     

湖泊微拟球藻高含油突变株的构建及筛选

研究旨在建立湖泊微拟球藻(Nannochloropsis)的遗传转化体系, 然后根据外源基因随机插入的特性, 挑取抗性转化子构建突变体库, 并从突变体库中筛选高含油突变株。以湖泊微拟球藻自身β-tublin基因启动子和三角褐指藻fcpA终止子驱动和终止来源于细菌的sh ble抗性选择基因, 构建了一个转化载体pPha-T1-TUB。将线性化后的质粒以电穿孔的方法转化湖泊微拟球藻, 通过1 μg/mL zeocin的抗性平板筛选, 并经液体培养基连续传代后, 得到了可以稳定遗传的转化子。我们从这些转化子中筛选得到了数株油含量高于野生型, 且生长也优于野生型的突变株。其中, 2个突变株K26和G5的总脂中多不饱和脂肪酸含量更低, 其脂肪酸组成更符合作为生物柴油原料的标准。研究通过随机插入构建突变体库的方法为快速获取优良目的性状的高产油突变株提供了一个有效手段。