圆红冬孢酵母磷酸盐饥饿诱导表达载体的构建

摘要:【目的】建立一种适用于圆红冬孢酵母代谢工程的磷酸盐饥饿诱导表达系统。【方法】对圆红冬孢酵母pho89基因5'侧翼序列进行生物信息学分析,设计相应引物,PCR扩增pho89基因启动子(pPHO89)和hsp70基因终止子(tHSP),利用RF克隆方法置换出发载体上的pPGK组成型启动子和tNOS终止子,以潮霉素磷酸转移酶基因hyg为报告基因,得到响应磷酸盐饥饿诱导的单表达盒载体pZPK-pPHO89-hyg-tHSP,利用ATMT方法转化圆红冬孢酵母,通过转化子潮霉素抗性表型鉴定pPHO89和tHSP的启动子和终止子活 性。在此基础上,构建了适合外源基因表达的双表达盒诱导表达载体pZPK-HYG-pPHO89-MCS-tHSP,并利用该载体构建了苹果酸酶重组表达菌株。【结果】成功构建了响应磷酸盐饥饿的圆红冬孢酵母诱导性表达载体,该载体在圆红冬孢酵母中可表现出启动子和终止子活性。【结论】该启动子受磷酸盐浓度的严谨调节,响应度高,操作简单,无需额外诱导剂,经济便捷,为后续圆红冬孢酵母代谢工程研究提供了基本材料。     

根癌农杆菌介导的灰葡萄孢菌遗传转化研究

以pCAMBIA1300-N载体为骨架, 成功构建了以绿色荧光蛋白(gfp)为报告基因, 潮霉素(hph)为抗性筛选标记的载体pKPG, 并利用根癌农杆菌介导转化系统, 成功获得了能表达绿色荧光蛋白的重组灰葡萄孢菌。通过PCR检测转化子的绿色荧光蛋白基因和潮霉素抗性表达框, 观察菌丝和分生孢子的荧光表型, 以及gfp基因的Southern杂交验证, 结果表明：被测转化子基因组中均成功整合了目的基因片段。     

农杆菌介导生防棘孢木霉菌转化体系的优化

目的:实现棘孢木霉菌T4的遗传转化并优化其转化体系.方法:以潮霉素抗性为选择标记,利用农杆菌转化法介导转化棘孢木霉菌.结果:潮霉素基因成功整合到受体菌基因组中,转化子抗性基因可稳定遗传.结论:最优的转化体系和条件为:IM和CM培养基中AS浓度为200 μg/mL,棘孢木霉T4孢子浓度为106/mL,农杆菌浓度为200 μL( OD600约0.8),共培养时间为48 h,转化效率约为50个转化子/106个孢子.     

双孢蘑菇萎锈灵抗性基因定点突变的载体构建与遗传转化

为建立更为安全、有效的双孢蘑菇遗传转化体系,构建了双孢蘑菇琥珀酸脱氢酶的铁硫蛋白亚基Agsdi1突变（His突变为Leu）表达载体pAgsdi1,并通过农杆菌介导方法转化双孢蘑菇W192,经萎锈灵筛选以及PCR扩增和MnlⅠ酶切验证后获得了转化菌株。验证结果表明,点突变的铁硫蛋白亚基Agsdi1可以作为双孢蘑菇有效的抗性标记基因。因其并未引入新的外源基因,是一种比潮霉素抗性基因更为安全的筛选标记,将可用于双孢蘑菇等食用菌的遗传转化。     

圆红冬孢酵母自主复制序列分离和利用游离型质粒进行基因敲除

【目的】与整合型表达载体相比,游离型表达载体通常具有更高的拷贝数以实现目标基因的高强度表达,并且对于DNA操作应用更加方便和灵活。然而,目前的研究尚未确定适用于圆红冬孢酵母的游离型质粒,该酵母外源基因的表达或者基于CRISPR/Cas9的基因组编辑都需要通过整合方式来完成,这也是对其遗传改造进展缓慢的一个重要原因。本研究目的是构建圆红冬孢酵母的游离型质粒,使得其外源基因的表达和基因组编辑更方便省时。【方法】首先对圆红冬孢酵母苯丙氨酸氨裂解酶基因(phenylalanine ammonia-lyase gene, PAL)中可能存在的自主复制序列(autonomously replicating sequences, ARSs)进行挖掘和表征,将该基因及其上下游序列进行分段扩增,构建到带有β-异丙基苹果酸脱氢酶基因(β-isopropyl malate dehydrogenase gene, LEU2)的质粒中,通过电转化的方法导入LEU2基因缺陷的圆红冬孢酵母中,根据转化效率高低鉴定了该酵母的一个ARS。其次,以编码香叶基香叶基焦磷酸合成酶(geranylgeranyl pyrophosphate synthase, GGPPS)的BTS1基因为敲除靶点,将其gRNA构建到基于ARS的游离型质粒中,通过转化子直观的颜色变化来验证该游离型质粒是否成功应用于圆红冬孢酵母的CRISPR/Cas9体系。【结果】本工作鉴定了圆红冬孢酵母的ARS,构建了基于ARS元件的游离型质粒,并将该质粒应用于圆红冬孢酵母CRISPR/Cas9体系,成功实现了基于游离型质粒的基因敲除。【结论】本研究丰富了圆红冬孢酵母现有的工具库,为圆红冬孢酵母的合成生物学应用提供了良好的研究基础和技术支持。     

根癌农杆菌介导转化红曲菌及转化子发酵性能的研究

利用根癌农杆菌介导转化技术成功将潮霉素抗性基因转入发白红曲菌中,优化了抗生素浓度,发白红曲菌孢子浓度,根癌农杆菌浓度,共培养温度及时间,以及乙酰丁香酮浓度等转化条件,最终转化效率可达52个转化子/105个红曲孢子.将转化子在含有潮霉素B的培养基继代培养5代,得到了多株稳定的转化子,对部分转化子进行PCR鉴定,结果进一步...     

红冬孢酵母分子遗传操作技术研究进展

红冬孢酵母(Rhodosporidium)是一种天然的微生物油脂和β-类胡萝卜素高产菌,有望被开发为工业平台菌株。近年来,基因组测序完成和各种组学研究的开展,为红冬孢酵母分子遗传操作技术开发奠定了基础。基因元件(如启动子、终止子、报告基因和筛选标记等)的挖掘和引入,基因转化方法开发以及基因操纵技术(如基因失活和过表达)等的完善,加速了红冬孢酵母的代谢工程研究进程。本文中,笔者总结了红冬孢酵母在分子遗传操作技术方面的研究进展,力图勾勒出红冬孢酵母的分子遗传操作技术研究的概貌,为后续研究提供借鉴。     

一种农杆菌介导的赤霉菌CCRC3.572的转化方法

目的:建立农杆菌Ti质粒介导的转化赤霉菌的新方法。方法:以农杆菌Ti质粒pCAMBIA0390为基础,构建带有潮霉素抗性基因表达盒的双元载体,并用农杆菌介导的方法转化赤霉菌。结果:构建了双元载体pCAMBIA0390-hph(PgpdA),并获得了具有潮霉素抗性的赤霉菌转化子。结论:农杆菌介导的方法适于赤霉菌的转化,为赤霉菌的遗传研究提供了一种新的手段。     

农杆菌介导的出芽短梗霉遗传转化及高效筛选聚苹果酸高产菌株

建立根癌农杆菌介导的出芽短梗霉遗传转化方法及T-DNA突变库,高效筛选聚苹果酸高产菌株及功能基因。通过含潮霉素和草铵磷抗性基因的农杆菌转化出芽短梗霉,抗性压力筛选及PCR验证建立根癌农杆菌介导的出芽短梗霉遗传转化方法,结合发酵液p H与聚苹果酸含量响应变化,微孔板高效筛选高产聚苹果酸的T-DNA插入突变株,基因组步移确定T-DNA插入位点及功能基因。结果获得遗传稳定的抗性基因菌株,每107个细胞可获得80-120个转化子,出芽短梗霉H27号T-DNA突变株聚苹果酸摇瓶发酵产量提高24.5%,基因组步移证实糖酵解途径磷酸甘油酸变位酶基因被破坏。成功建立了根癌农杆菌介导的出芽短梗霉遗传转化方法和T-DNA插入突变库,结合高效筛选方法为聚苹果酸合成功能基因挖掘及高产机制解析奠定基础。     

双孢蘑菇耐热相关基因的表达载体构建及转化研究

构建了双孢蘑菇Agaricus bisporus耐热相关基因028-1全长cDNA序列的双元表达载体,通过农杆菌介导转化双孢蘑菇非耐热菌株8213,经潮霉素抗性筛选和PCR鉴定,获得了一批双孢蘑菇转基因菌株。对10株转基因菌株进行了不同温度下的草管走菌试验,结果显示大部分转基因菌株的耐热性能有较为明显的提高。     

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.