低双乙酰抗老化啤酒酵母工程菌的构建

用来源于啤酒酵母的γ-谷氨酰半胱氨酸合成酶基因（GSH1）和铜抗性基因（CUP1）取代质粒pLZ-2中α-乙酰乳酸合成酶基因（ILV2）内部约2.3kb的DNA片段,构建成重组质粒pICG。限制酶酶切质粒pICG后获得在基因GSH1和CUP1两端含有ILV2序列的6.0kb的DNA片段。用此片段转化啤酒酵母YSF31,得到铜抗性高的转化子。并通过PCR和α-乙酰乳酸合成酶（AHAS）活性测定筛选到酵母工程菌。小试实验结果表明酵母工程菌谷胱甘肽含量比受体高34％,而双乙酰含量是受体的75％。其他发酵指标并没有发生改变。中试实验表明酵母工程菌发酵周期缩短3d,而且成品啤酒的保鲜时间延长50％。由于DNA操作过程中没有外源基因介入,因此啤酒酵母工程菌为生物安全的自克隆菌株,具有重要的实际应用价值。     

甾醇酰基转移酶基因高表达对酵母菌麦角甾醇合成的影响

通过PCR扩增克隆到含酵母菌甾醇酰基转移酶基因ARE2编码序列和上游调控序列的DNA片段ARE21及仅含编码序列的DNA片段ARE22。分别以ARE2启动子,乙醇脱氢酶基因ADH1启动子和铜抗性基因CUP1启动子及ADH1终止子为调控元件构建了酵母菌表达质粒pHX2,pHXA2和pHXC2。表达质粒分别转化酿酒酵母单倍体菌株YS58和以前通过细胞杂交构建的麦角甾醇高产菌株YEH56。通过营养缺陷互补和铜抗性筛选到转化子,质粒上的ARE2基因在YS58和YEH56中都实现了活性表达,使细胞内甾醇酯化水平升高,并导致细胞麦角甾醇含量的提高。对转化菌株的培养条件进行了初步研究,在优化条件下,重组转化菌株YEH56(pHX2)、YEH56(pHXA2)和YEH56(pHXC2)的麦角甾醇含量分别是受体菌YEH56 的13、13和14倍。     

构建高产谷胱甘肽啤酒酵母基因工程菌提高啤酒抗老化能力的研究

根据同源重组的原理,将来源于啤酒酵母工业菌株G03的γ-谷氨酰半胱氨酸合成酶基因(GSH1)和筛选标记Kan取代质粒pRJ-5中18S rDNA内部约340bp的DNA片段,构建重组质粒pRKG。以pRKG为模版,PCR得到以18S rDNA为整合位点包含GSH1和Kan的基因片段18S rDNA::(Kan-GSH1)。用此片段转化啤酒酵母工业菌株G03,通过G418抗性筛选得到啤酒酵母工程菌。实验室小试表明,工程菌的谷胱甘肽含量比受体菌株提高16.6%,啤酒的抗老化能力得到了显著提高,而常规指标没有发生显著变化。连续传代5次后胞内GSH含量基本不变遗传稳定性良好。由于表达γ-谷氨酰半胱氨酸合成酶的基因来源于受体菌株自身,是通过自克隆技术改造工业啤酒酵母的一次有益的尝试。     

构建高产谷胱甘肽啤酒酵母基因工程菌提高啤酒抗老化能力的研究

根据同源重组的原理,将来源于啤酒酵母工业菌株G03的γ-谷氨酰半胱氨酸合成酶基因（GSH1）和筛选标记Kan取代质粒pRJ-5中18S rDNA内部约340bp的DNA片段,构建重组质粒pRKG。以pRKG为模版,PCR得到以18S rDNA为整合位点包含GSH1和Kan的基因片段18S rDNA::(Kan-GSH1)。用此片段转化啤酒酵母工业菌株G03,通过G418抗性筛选得到啤酒酵母工程菌。实验室小试表明,工程菌的谷胱甘肽含量比受体菌株提高16.6%,啤酒的抗老化能力得到了显著提高,而常规指标没有发生显著变化。连续传代5次后胞内GSH含量基本不变遗传稳定性良好。由于表达γ-谷氨酰半胱氨酸合成酶的基因来源于受体菌株自身,是通过自克隆技术改造工业啤酒酵母的一次有益的尝试。     

敲除啤酒酵母 fks1 基因对啤酒酵母自溶性能的影响

以啤酒酵母G-03为模板,扩增得到铜抗性基因(cup 1)和β-葡聚糖合成酶基因(fks 1)。将fks 1连接pMD-18T Vector得到重组质粒pTK,重组质粒pTK和cup1经Bgl Ⅱ、Sal Ⅰ酶切后连接得到重组质粒pKC。Bam HI酶切重组质粒pKC得到以fks 1为整合位点包含cup1的基因片段fks 1::cup1。用此片段转化啤酒酵母工业菌株G-03,通过硫酸铜抗性筛选得到一株啤酒酵母工程菌G-03/C。工程菌连续传代10次后依然能在筛选平板上生长,遗传稳定性良好。主酵结束G-03/C和G-03的辛酸和癸酸含量基本相同。25℃诱导自溶20 d,G-03/C的辛酸、癸酸分别下降57.3%、81.8%,自溶性能减弱。G-03/C的死亡率、双乙酰、浊度及TBA均较原菌有所下降。G-03/C与G-03酿制成品啤酒的常规指标没有较大差别,品评结果表明,G-03/C风味更优。     

利用五碳糖产高纯度L-乳酸的大肠杆菌基因工程菌的构建

[目的]本研究以已敲除多个产杂酸酶基因的大肠杆菌(Escherichia coli)乙醇工程菌SZ470(△frdBC △ldhA △ackA △focA-pflB △pdhR::pflBp6-pflBrbs-aceEF-lpd)为起始菌株,进一步敲除其乙醇脱氢酶(alcohol dehydrogenase,ADH)基因,同时插入带有自身启动子的乳酸片球菌(Pediococcus acidilactici)的L-乳酸脱氢酶(L-lactate dehydrogenase,LLDH)基因,构建可利用五碳糖同型发酵L-乳酸重组大肠杆菌.[方法]利用λ噬菌体Red重组系统构建乙醇脱氢酶基因(adhE)缺失菌株Escherichia coli JH01,并克隆P.acidilactici的ldhL基因,利用染色体插入技术将其整合到JH01基因组,构建产L-乳酸大肠杆菌基因工程菌Escherichia coli JH12,利用无氧发酵15 L发酵罐测定重组菌株L-乳酸产量.[结果]工程菌JH12在15 L发酵罐中以6％的葡萄糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为1.46 g/(L·h),乳酸生产强度为1.14 g/(L·h),乳酸的产量达到41.13 g/L.发酵产物中未检测到琥珀酸、甲酸的生成,仅有少量乙酸生成,L-乳酸纯度达95.69％(L-乳酸在总发酵产物的比率).工程菌JH12以6％的木糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为0.88 g/(L·h),乳酸生产强度为0.60 g/(L·h),乳酸的产量达到34.73 g/L.发酵产物中杂酸少,乳酸的纯度高达98％.[结论]本研究通过基因敲除、染色体插入及无氧进化筛选获得一株产L-乳酸的大肠杆菌工程菌JH12,该菌株不需利用外源质粒,稳定性好,可利用五碳糖进行发酵,发酵产物中杂酸少,L-乳酸的纯度高.本研究为L-乳酸大肠杆菌工程菌的构建提供一定的技术支持,同时也为大肠杆菌L-乳酸的工业化生产提供了参考依据.     

过表达INO1基因提高酿酒酵母乙醇耐受性

为获得燃料乙醇生产菌株,通过基因工程改造,构建能够利用能源甘蔗汁发酵、乙醇产率高的酿酒酵母工程菌株。即过表达肌醇-3-磷酸合成酶基因ino1,敲除kanMX抗性基因,获得重组菌。对过表达菌株的乙醇耐受性进行分析。利用甘蔗汁进行发酵培养,采用气相色谱(GC)对发酵产物乙醇进行检测。结果显示过表达菌株YI2-1能够耐19%(V/V)的乙醇,利用20oBx甘蔗汁厌氧发酵乙醇积累量为13.10%(V/V),较出发菌提高了8.55%。而过表达菌株YI2-1△KP的最大乙醇积累量为13.17%(V/V),较出发菌提高了9.16%。研究表明通过过表达酿酒酵母ino1基因能够有效提高菌株细胞活力、乙醇耐受性。构建的工程菌可利用甘蔗汁发酵,具有较高的乙醇产量。     

黑曲霉木聚糖酶在工业酒精酵母中的分泌表达

用重叠延伸PCR方法从黑曲霉 (Aspergillusniger)UV 11的基因组DNA中克隆出木聚糖酶的cDNA基因 ,构建了由酵母乙醇脱氢酶 (ADH1)启动子和终止子引导表达、木聚糖酶自身信号肽引导分泌、rDNA序列介导的酵母整合型分泌表达质粒pAX2。用pAX2与酵母YEp型G4 18抗性质粒共转化野生型工业酒精酵母S .cerevisiae 2 346 ,获得了整合型分泌表达木聚糖酶的酵母重组菌株XY2。发酵分析表明该工程菌能够明显提高酒精生产率     

酿酒酵母乙醇脱氢酶2(ADH2)基因的克隆表达及活性验证

为了从酿酒酵母Saccharomyces cerevisiae中克隆出乙醇脱氢酶2(Alcoholdehy drogenase2,ADH2)基因并使之在大肠杆菌中高效表达。以酿酒酵母细胞中提取的总RNA为模板,通过反转录获得酿酒酵母乙醇脱氢酶2基因,连接到表达载体pTAT上,得到重组表达质粒pTAT-ADH2,将此重组质粒转化到大肠杆菌BL21中,重组工程菌株经IPTG诱导表达得到ADH2蛋白。将该蛋白纯化后,在体外进行活性检测和小鼠体内进行毒理试验,检测ADH2的酶活性。测序结果表明克隆的基因与GenBank中所报道的adh2基因序列有90%的同源性,经SDS-PAGE电泳分析,目的蛋白得到了有效表达,蛋白条带扫描分析表明,表达量占总蛋白的50%左右,纯化得到的蛋白在小鼠体内进行毒理试验,显示出一定的活性。酿酒酵母adh2基因的克隆正确,不仅在大肠杆菌中进行了高效表达而且表现出了较好的酶活性。     

米根霉乙醇脱氢酶(ADH)突变菌株的诱变选育

米根霉发酵生产L-乳酸过程中,由于丙酮酸在丙酮酸脱羧酶、乙醇脱氢酶(ADH)催化下生成乙醇,使得丙酮酸向乳酸转化的流量减少。采用亚硝基胍(NTG)诱变米根霉AS3．3462孢子液,诱变剂量为0．15 mg/ mL时,致死率为70%～80%。在含丙烯醇的YPD筛选培养基上筛选获得两株ADH活力降低的突变株mut-1和mut-2,检测突变株mut-1和mut-2的最大ADH活力分别为35．67和43．09U/mL,是原始菌株的41．63%和50．29%。发酵72h后,原始菌株的乙醇与乳酸浓度分别为28．9g/L和40．31g/L,而mut-1和mut-2突变株的乙醇产量分别为4．87g/L和6．56g/L,乳酸产量为54．45g/L和44．07g/L。在相同的发酵条件下,米根霉ADH突变株mut-1和mut-2对还原糖的利用速率高于出发菌株,其生物量积累亦高于出发菌株。     

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.