卷枝毛霉Δ6-脂肪酸脱氢酶基因的克隆及在酿酒酵母中的高效表达

为获得产高γ 亚麻酸的酿酒酵母工程菌株,应用RT PCR技术,从卷枝毛霉中扩增出△6 脂肪酸脱氢酶 基因,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒 pYES412,用醋酸锂方法转化到酿酒酵母缺陷型菌株INVScI中,在SC ura合成培养基中筛选到转化酵母,在合适 的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体,通过气相色谱对转化酵母进行脂肪酸 色谱分析,结果表明:γ 亚麻酸占总脂肪的50.07%。迄今为止,这是国内外△6 脂肪酸脱氢酶基因在酿酒酵母表 达量最高的报道。     

卷枝毛霉△^6—脂肪酸脱氢酶基因的克隆及在酿酒酵母中的高效表达

为获得产高γ—亚麻酸的酿酒酵母工程菌株,应用RT—PCR技术,从卷枝毛霉中扩增出△^6—脂肪酸脱氢酶基因,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2．0,在大肠杆菌中筛选到含有目的基因的重组质粒pYES412,用醋酸锂方法转化到酿酒酵母缺陷型菌株INVScl中,在SC—ura合成培养基中筛选到转化酵母,在合适的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体,通过气相色谱对转化酵母进行脂肪酸色谱分析,结果表明：γ—亚麻酸占总脂肪的50．07％。迄今为止,这是国内外△^6—脂肪酸脱氢酶基因在酿酒酵母表达量最高的报道。     

玉米△12脂肪酸脱氢酶基因（FAD2）在酿酒酵母中的表达

玉米△12脂肪酸脱氢酶是催化油酸形成亚油酸的关键酶。将其编码基因FAD2（GenBank登陆号：DQ496227）克隆到酿酒酵母表达载体pYES2．0中,构建成重组质粒pYE／FAD2,转化到酿酒酵母进行诱导表达,同时以pYES2．0转化子为对照。气相色谱（Gc）分析表明,重组转化子亚油酸的含量占酵母总脂肪酸的1．54％,而对照未检测到亚油酸。表明FAD2基因具有编码△12脂肪酸脱氢酶的功能。为探索转译起始密码子周边序列的改变对FAD2基因表达产生的影响,将该基因的起始密码子上游序列进行修改,构建重组表达载体pYE／FAD2—1,转化酿酒酵母进行表达。GC分析表明,pYE／FAD2—1转化子的亚油酸含量占总脂肪酸含量的8．81％,是对照pYE／FAD2转化子的近5倍。     

花生Δ~(12)脂肪酸脱氢酶基因高效表达载体构建与原核表达(英文)

花生是世界范围内广泛种植的重要油料作物之一,其种子中富含油酸和亚油酸。△12脂肪酸脱氢酶(FAD2)是亚油酸合成的关键酶,催化油酸(18:1)在△12位上脱氢生成亚油酸(18:2),但由于△12脂肪酸脱氢酶本身的特性,目前还没有有效的方法将其纯化并在蛋白水平作进一步的研究,尚需对其结构和功能之间以及表达调控进行更深入全面的研究。本文利用从花生中克隆的△12脂肪酸脱氢酶基因(GenBank接受号为AY1006)构建高效表达载体,把花生?12脂肪酸脱氢酶基因全长序列插入到大肠杆菌高效表达载体pRSETB中,构建了pRSET/HO-A融合表达载体,并转化到大肠杆菌表达菌BL21(DE3)pLysS中,在IPTG诱导下,pRSET/HO-A融合表达载体在BL21(DE3)pLysS菌株中高效表达了?12脂肪酸脱氢酶。利用Clon-Tech蛋白纯化Kit进一步分离了目的蛋白,同时加入外源性底物油酸在20℃温育6h后,进行脂肪酸甲酯化处理,通过气相色谱(GC)和气相色谱/质谱(GC-MS)分析表明,所编码的酶具有?12脂肪酸脱氢酶的活性,能将外源性的底物油酸转化为亚油酸,转化率为11.8%。花生△12脂肪酸脱氢酶基因的原核表达目前国内外还未见报导,本实验为其进一步的大量纯化和结构功能分析奠定了基础。     

转译起始密码子周边序列的改变对Δ6-脂肪酸脱氢酶基因表达的影响

将少根根霉中Δ6-脂肪酸脱氢酶基因(RAD6)的起始密码子周边序列作适当的修改,并把修改后获得的片段(RAD6_1)亚克隆到表达载体pYES2.0,构建重组表达载体pYRAD6_1。经测序验证,把pYRAD6_1转化到酿酒酵母的缺陷型菌株INVScl进行表达分析,同时以空载体pYES20和出发序列所构建的pYRAD6作为对照。通过气相色谱(GC)和气相色谱/质谱(GC_MS)分析表明,在pYRAD6和pYRAD6_1转化的酿酒酵母中生成γ_亚麻酸,而pYES2.0中没有检测到。其中,pYRAD6-1转化的酿酒酵母γ_亚麻酸表达量占细胞总脂肪酸含量的5.23%,而对照pYRAD6转化的酿酒酵母中表达量只占2.64%。     

高山被孢霉ATCC16266Δ~6-脂肪酸脱氢酶基因在酿酒酵母中的表达

Δ6 脂肪酸脱氢酶是形成γ 亚麻酸的关键酶。从含有高山被孢霉Δ6 脂肪酸脱氢酶基因的重组质粒pT MACL6中 ,酶切出 1 4kb的目的片段 ,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体 pYES2 .0 ,在大肠杆菌中筛选到含有目的基因的重组质粒 pYMAD6 ,用醋酸锂方法转化到酿酒酵母的缺陷型菌株INCSc1中 ,在SC Ura合成培养基中 ,选择得到酿酒酵母工程株YMAD6。在合适的培养基及培养条件下 ,加入外源底物亚油酸 ,经半乳糖诱导后 ,收集菌体。通过GC MS对酵母工程株进行脂肪酸色谱分析 ,结果表明 ,产生了 31 6 %的γ 亚麻酸。这是迄今为止 ,国内外Δ6 脂肪酸脱氢酶基因在酿酒酵母中表达量最高的报道。     

一种新型酿酒酵母附加型分泌表达载体的构建

用化学法合成克鲁维酵母的菊粉酶基因的信号肽序列(INU),将其嵌入酵母附加型表达质粒pYES2,得到一套新型的分泌表达载体pYES2I,pYES2Ⅱ,pYES2Ⅲ。然后用PCR方法分别扩增大肠杆菌的天冬酰胺酶基因(ASN)和短芽孢杆菌α乙酰乳酸脱羧酶(ALDC)基因,连接到INU下游,得到重组质粒pASN和pALDC。分别将这两个重组质粒转化酿酒酵母菌株INVScⅠ中表达,胞内和胞外的酶活分析表明ASN和ALDC基因都能在酿酒酵母中分泌表达,表明菊粉酶信号肽序列能很好地将酿酒酵母中的重组蛋白分泌到胞外。稳定性分析表明,重组酵母菌株在没有选择压力的条件下连续接种培养100h,未发现重组质粒的不稳定性。     

高山被孢霉ATCC16266△^6—脂肪酸脱氢酶基因在酿酒酵母中的表达

△^6－脂肪酸脱氢酶基因是形成γ－亚麻酸的关键酶。从含有高山被孢霉△^6－脂肪酸脱氢酶基因的重组质粒pT-MACL6中,酶切出1.4kb的目的片段,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒pYMAD6,用醋酸昔方法转化到酿洒酵母的缺陷型菌株INCSc1中,在SC－Ura合成培养基中,选择得到酿酒酵母工程株YMAD6。在合适的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体。通过GC－MS对酵母工程株进行脂肪酸色谱分析,结果表明,产生了31.6％的γ－亚麻酸,边是迄今为止,国内外△^6－脂肪酸脱氢酶基因在酿酒酵母中表达量最高的报道。     

代谢木糖和葡萄糖的重组酿酒酵母的构建*

为使酿酒酵母(Saccharomyces cerevisiae)YS58代谢木糖产乙醇,采用PCR方法克隆得到树干毕赤酵母(Pichia stipitis)木糖醇脱氢酶基因xyl2,并将该基因和克隆得到的休哈塔假丝酵母(Candida shehatae)缺终止子的木糖还原酶基因xyt1一起连接到酵母表达载体pYES2的强启动子GAL下,得到融合表达载体pYES2-P12。通过醋酸锂转化的方法将pY- ES2-P12转入S．Cerevisiae YS5     

转译起始密码子周边序列的改变对△^6-脂肪酸脱氢酶基因表达的影响

将少根根霉中△^6-脂肪酸脱氢酶基因(RAD6)的起始密码子周边序列作适当的修改,并把修改后获得的片段(RAD6-1)亚克隆到表达载体pYES2．0,构建重组表达载体pYRAD6-1。经测序验证,把pYRAD6-1转化到酿酒酵母的缺陷型菌株INVScl进行表达分析,同时以空载体pYES2．0和出发序列所构建的pYRAD6作为对照。通过气相色谱(GC)和气相色谱,质谱(GC-MS)分析表明,在pYRAD6和pYRAD6-1转化的酿酒酵母中生成γ-亚麻酸,而pYES2．0中没有检测到。其中,pYRAD6-1转化的酿酒酵母γ-亚麻酸表达量占细胞总脂肪酸含量的5．23％,而对照pYRAD6转化的酿酒酵母中表达量只占2．64％。     

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.