高SO2产量啤酒酵母工业菌株的构建

二氧化硫在啤酒中具有抗氧化的重要功能,而在其形成过程中APS激酶(MET14编码)起着非常重要的作用。以二氧化硫产量较高的青岛啤酒酵母(Saccharomyces cerevisiae)YSF-5的总DNA为模板,用PCR方法克隆得到MET14基因。为使目的基因在酿酒酵母中表达,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以PGK1强启动子为调控元件,构建了重组表达质粒pPM,并转化酿酒酵母YS58。转化子在YNB添加亮氨酸、组氨酸和色氨酸的选择性培养基上筛选鉴定,盐酸副玫瑰苯胺法测得转化子的SO2产量是受体菌的2倍左右。在重组表达质粒pPM的基础上添加铜抗性标记基因构建了重组表达质粒pCPM,并转化青岛啤酒工业酵母菌株YSF-38,转化子在YEPD 4mmol/L CuSO4的选择性培养基上筛选鉴定,实验室条件下培养后,测得转化子YSF-38(pCPM)的SO2产量是受体菌的3.2倍。用该转化子在青岛啤酒厂进行小型发酵实验,结果表明在发酵结束时,YSF-38(pCPM)转化子的SO2产量是受体菌的1.4倍。因此,MET14基因的有效表达可以提高啤酒工业酵母的SO2产量。     

产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因（CgGPD）功能分析

【目的】从高产甘油生产菌株产甘油假丝酵母（Candida glycerinogenes）基因组中克隆了NAD+依赖3-磷酸甘油脱氢酶编码基因（CgGPD）,但是该基因及其上游调控序列具体的功能还是未知的。本文研究了CgGPD基因及其上游调控序列的功能。【方法】本文以酿酒酵母（Saccharomyces cerevisiae）及其渗透压敏感型突变株为宿主,构建3种不同的酵母表达载体导入酵母细胞,研究了不同酵母转化子在渗透压胁迫条件下CgGPD基因表达对细胞的耐高渗透压胁迫应答及其细胞的甘油合成能力的影响。【结果】实验结果表明无论是以来源于S. cerevisiae 的TPI启动子还是来源于CgGPD基因的启动子,过量表达CgGPD基因的转化子均能够显著加速葡萄糖消耗速度和提高甘油合成能力,在gpd1/gpd2突变株中表达CgGPD基因能够消除细胞对外界高渗透压的敏感性,同时转化子胞内甘油大量积累。【结论】CgGPD基因在野生型酵母S. cerevisiae W303-1A表达显著提高细胞的甘油合成能力,在gpd/1gpd2突变株中能够互补GPD1基因的功能,CgGPD基因表达受渗透压诱导 调控。     

几株啤酒酵母的筛选及发酵性能比较*

啤酒酵母是啤酒酿造的灵魂,可以直接影响啤酒品质。在啤酒酿造过程中,由于啤酒酵母被多次传代和保藏,造成优良菌种发酵性能衰退等问题,导致发酵不彻底,影响最后啤酒的风味质量。为此以8株Lager型啤酒酵母为出发菌株,通过平板分离纯化获得80株分离菌株,再经过三角瓶发酵初筛和复筛、发酵罐中试发酵实验最终获得了8株发酵性能优良的啤酒酵母。其中,6株酵母可应用于酿造双乙酰含量低于0.1 mg/L的啤酒;3株酵母发酵度高于70%,适合酿造干啤酒;1株酵母发酵度低于50%,适合酿造低醇啤酒。在风味方面:1株酵母酿造的啤酒醇酯比为3.3,啤酒酯香味较突出;另1株酵母酿造的啤酒醇酯比为4.5,啤酒高级醇含量较高。8株经过选育的啤酒酵母发酵特征明显,便于精酿啤酒厂实际应用。     

ILV2基因缺失对啤酒酵母生长与双乙酰代谢的影响

【目的】旨在应用分子生物学方法降低啤酒发酵液中双乙酰含量,改善啤酒感官质量。【方法】以酿酒酵母S2(Saccharomyces cerevisiae)为出发菌株,通过同源重组敲除四倍体啤酒酵母α-乙酰乳酸合成酶部分基因(ILV2),构建缺失一个和两个ILV2等位基因的突变株QI2-1和QI2-2,并进行啤酒发酵实验。【结果】ILV2基因的缺失,会导致菌株初始生长速率的降低。其中QI2-2较为明显,12 h时,突变株与出发菌株的生长速率达到一致。啤酒发酵结果表明,与出发菌株相比,突变株QI2-1双乙酰峰值与双乙酰最终含量分别降低17.50%和17.83%,而QI2-2分别降低51.67%和45.65%。其他啤酒指标如酒精度、发酵度、残糖和风味物质等略有变化,但都在优质啤酒指标范围内,符合啤酒发酵的质量要求。【结论】通过同源重组敲除部分ILV2基因和选育低产双乙酰菌株是降低啤酒双乙酰含量、提高啤酒质量的有效方法,具有一定的实际应用价值。     

mal62 基因高表达对工业面包酵母发酵力的影响

【目的】构建高麦芽糖利用能力的面包酵母菌株,以期提高面包酵母在不加糖面团中的发酵力,增加经济效益的同时减少成本消耗。【方法】克隆工业面包酵母BY-14的麦芽糖酶基因mal62,以PGK1强启动子和终止子为调控元件,以酵母-大肠穿梭型质粒Yep-C为载体,构建重组表达质粒Yep-CPM,并转化酿酒酵母(Saccharomyces cerevisiae)BY-14,经筛选鉴定获得酵母转化子BYCPM。进行转化子的酶活力、mal62基因表达水平及发酵力测定,检测目的基因的功能性表达。【结果】工业酵母转化子BYCPM的最大麦芽糖酶活力比对照菌提高15%-52%,发酵力提高40%,比发酵力提高5.6%。【结论】转化子BYCPM具有更高的麦芽糖酶活力和更强的抗葡萄糖阻遏能力。并且在不加糖面团中,转化子具有更高的发酵力,可以在更短的时间内获得更大的产气量且消耗更少的碳源。     

用带有α-乙酰乳酸脱羧酶编码基因的固定化酵母生产啤酒

啤酒酵母VTT-A-88085(A-85)是含有α-乙酰乳酸脱羧酶(α-ALDC)基因的试验菌株。它是由深层发酵啤酒酵母VTT-A-63015(A-15)菌株构建的转化子。A-85是一株整合菌昧,即α-ald基因插入到酵母染色体DNA上,因此比用质粒转化得到的转化子更稳定。转化子产生的α-ALDC酶可直接把α-乙酰乳酸转化成乙偶姻而不形成双乙酰,因此可以免除或缩短常规啤酒发酵所需的冗长的后期发酵。     

2型猪链球菌SSU0448基因敲除突变株的构建及其毒力分析

【目的】 通过构建2型猪链球菌(SS2)强毒株05ZYH33的SSU0448基因缺失突变株Δ0448和互补株CΔ0448,探索SSU0448基因缺失对细菌基本生物学特性和细菌毒力的影响。【方法】用同源重组基因敲除方法构建筛选强毒株05ZYH33中N-乙酰半乳糖胺和半乳糖胺代谢途径相关转录调节因子SSU0448基因的缺失突变株,比较分析突变株Δ0448与野生株05ZYH33、互补株CΔ0448的基本生物学特性,小鼠毒力实验分析SSU0448基因缺失对细菌毒力的影响。【结果】PCR检测分析显示,SSU0448基因在转化重组体中被壮观霉素抗性基因所替代,表明基因敲除突变株构建成功;同时构建了基因功能互补株CΔ0448。生物学特性实验表明突变株Δ0448在成链能力上较野生株明显减弱,对数生长期稍短,快速到达平台期;而菌落形态、革兰氏染色和溶血活性方面无明显差异;小鼠毒力实验发现,突变株毒力并无显著改变。【结论】SSU0448基因的敲除能够改变2型猪链球菌的成链能力;不影响其侵袭致病能力,可能延缓2型猪链球菌的发病过程,此研究为2型猪链球菌致病感染奠定了基础。     

高致病性2型猪链球菌截短型类枯草杆菌丝氨酸蛋白酶基因的鉴定和功能研究

【目的】克隆表达高致病性2型猪链球菌05ZYH33株的SspA截短型基因,验证其是否具有酶学活性,并构建该基因的缺失突变株细菌,探讨其在2型猪链球菌致病过程中所起的作用【。方法】构建SS2的SspA截短型基因05SSU0811原核表达质粒,表达并纯化05SSU0811蛋白,运用丝氨酸蛋白酶底物Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide(pNa),通过显色反应检测表达产物的酶学活性;运用同源重组技术敲除05SSU0811基因,多重交叉PCR筛选敲除株并测序鉴定,动物试验分析05SSU0811基因缺失对细菌毒力的影响。【结果】成功表达并纯化05SSU0811蛋白,浓度约为3.5 g/L。丝氨酸蛋白酶活性测定试验证实其具有酶学活性;获得05SSU0811基因缺失突变株,小鼠攻毒试验表明,野生株攻毒的20只小鼠全部死亡,基因缺失突变株攻毒组死亡9只,死亡率45%,两组间死亡率有显著性差异。表明05SSU0811基因缺失的菌株毒力较野生株明显下降。【结论】05SSU0811基因编码的截短型丝氨酸蛋白酶仍然具有酶学活性,SS2的截短型基因SspA在高致病性2型猪链球菌的致病性方面具有一定作用。     

啤酒酵母代谢工程研究进展

啤酒工业上应用的啤酒酵母菌株在生产中都会存在着某些方面的缺陷。通过分析啤酒酵母某些代谢产物的代谢途径,寻找改变其代谢流量的方法,然后用分子生物学手段对其代谢流量加以改变,来调节啤酒酵母某些产物的代谢水平已经成为啤酒酵母育种的新方式。对酵母的底物利用、可操作性、控制有害副产物的产量及改善啤酒风味等方面的研究成果进行了综述。     

海水养殖生境中柠檬酸杆菌降解半胱氨酸产生硫化物的机制研究

【目的】含硫氨基酸的生物降解是造成海水养殖环境中毒性硫化物上升的重要因素,而微生物降解含硫氨基酸的机制和影响因素解析是控制该系统中硫化物浓度的关键环节。【方法】本研究利用稀释涂布-叠皿夹法自本实验室海水养殖环境的沉积物中分离得到一株产生硫化物的厌氧菌株,并通过代谢组学研究其以半胱氨酸为底物产生硫化物的机制和途径。【结果】经鉴定,该菌株为弗氏柠檬酸杆菌(Citrobacter freundii),能够在厌氧条件下还原硫酸盐,能够降解半胱氨酸产生硫化物,投加L-半胱氨酸可提高其还原硫酸盐的能力。该菌株以1 g/L半胱氨酸为底物,在35℃、盐度为10、pH 8.0的条件下,硫化物的最高积累量可达302.4 mg/L。对菌株中硫化物产生具有重要贡献的半胱氨酸脱巯基酶研究表明,该酶最适温度为35℃,在pH 6.0–8.0有较高的活性,能够快速降解半胱氨酸产生硫化物。结合代谢组学研究发现,该菌株中含有的3-巯基丙酮酸硫酸转移酶、胱硫醚-γ-裂解酶、半胱氨酸脱巯基酶催化半胱氨酸降解是产生硫化氢的主要途径;亚硫酸盐还原酶还原硫酸盐、亚硫酸盐是其产生硫化氢的次要途径。【结论】通过揭示柠檬酸杆菌降解半胱氨酸产生硫化物的机理,可为海水养殖环境中毒性硫化物的防控提供理论基础。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.