基于Golden Gate技术的齐整小核菌转录单元高效组装系统

【目的】为齐整小核菌代谢工程研究建立高效的转录单元组装系统。【方法】通过应用Golden Gate技术,以mobius assembly为基础,分别设计并构建DNA元件标准化接口改造、单转录单元组装、应用质粒(多转录单元)组装等功能的载体,从而形成一套完整的多转录单元组装系统。【结果】构建了2个用于DNA元件标准化接口改造的Level 0载体,4个用于单转录单元组装的Level 1载体,4个用于应用质粒组装的Level 2载体和13个应用质粒组装的辅助质粒。然后应用此系统为齐整小核菌组装了若干经过标准化接口改造的DNA元件质粒、单转录单元质粒和硬葡聚糖相关基因的功能分析质粒。所构建的最终应用质粒可以同时适用于齐整小核菌的根癌农杆菌介导转化法、电穿孔转化法和原生质体转化法。【结论】此质粒系统具有强大的DNA设计、组装和容纳能力,为未来齐整小核菌代谢工程和功能基因组学研究提供了高效的质粒构建技术平台。     

发酵法生产小核菌多糖

本文报道小核菌多糖产生菌株的筛选及一株齐整小核菌(Sclerotium rolfsii Sacc.)No.1产生胞外多糖的条件。在16升自控罐发酵试验中,多糖产量达14.14g/l,多糖对底物的转化率为47.52%。     

电穿孔法转化齐整小核菌

【目的】建立齐整小核菌(Sclerotium rolfsii)的电穿孔法转化方案,以实现经济、快速地遗传转化。【方法】将齐整小核菌gpd基因启动子控制的basta抗性基因bar与红色荧光蛋白基因DsRed Max组成的融合蛋白表达盒,通过电击转入野生型齐整小核菌细胞中,筛选转化子并进行PCR与荧光观察验证。在此基础上,测试了不同电压、脉冲时间、外源DNA片段与受体细胞比例等条件下的转化效率,以得到优化的电转化参数。最后,采用优化的条件,尝试转化多种抗性基因与荧光蛋白融合表达盒以测试其可用性。【结果】成功得到了bar、sdhR与aphI基因的转化子。【结论】成功建立了优化的齐整小核菌电穿孔转化法。优化的参数条件为电压2kV/cm、脉冲时间1 ms、DNA/匀浆细胞比例3μg/300 mg,电击1次。     

油茶白绢病原菌齐整小核菌分子检测的研究

目的：设计特异性引物建立油茶白绢病齐整小核菌的快速分子检测体系。方法：扩增齐整小核菌核糖体DNA ITS区并测定其序列,比较该序列与GenBank中近似种的ITS序列差异,设计了特异性引物BF1和BR2。结果：该引物可以从齐整小核菌中扩增得到约540bp特异性条带,而扩增其它近似或相关菌株时没有相应的特异性条带。在25μL PCR反应体系中,引物BF1和BR2检测灵敏度为1pg浓度DNA。结论：利用设计的BF1和BR2特异性引物结合PCR方法可快速的扩增出齐整小核菌DNA,检测灵敏度为1pg.但在生产实践中诊断油茶白绢病发病前组织中的齐整小核菌还需要进一步研究。     

丝核菌的分类系统：现状及存在问题

以重要植物病原菌为特征的丝核菌是一类在土壤中广泛分布的丝状真菌,通常不产孢,以菌丝或菌核的形式存在,多样性非常丰富。本文基于国内外最新研究进展,对依据菌丝体的细胞核数目、菌丝融合、有性生殖和系统进化等方面的基本特征展开的丝核菌分类体系及分类现状进行了综述。基于菌丝的细胞核数目,丝核菌被分为单核、双核和多核丝核菌三大类群。自然界中单核丝核菌数量极少,多核和双核丝核菌在全球分布广泛,占丝核菌的绝大多数。基于菌丝融合试验的结果,目前多核丝核菌被分为13个菌丝融合群,双核丝核菌被分为18个菌丝融合群。部分融合群内又根据一些稳定的特征分了亚群,但亚群的建立标准并不统一。目前的分子系统学研究结果基本支持丝核菌的菌丝融合群及亚群的分类。基于部分有性世代被发现的菌株的形态特征,多核和双核丝核菌分别被鉴定为亡革菌属和角担菌属。此外,目前已有分属重要植物病原菌和兰科菌根菌类群的至少9个融合群或亚群的17个菌株完成了基因组测序,比较基因组学和线粒体组学开始在丝核菌分类和进化研究中发挥作用。丝核菌分类系统特殊且复杂,作者在文末提出了目前丝核菌分类学研究面临的问题和今后研究的趋势,期待更多的学者参与到这个重要菌...     

立枯丝核菌遗传多样性研究方法介绍

立枯丝核菌（Rhizoctonia solani Kühn）是一个集合种，遗传多样性丰富.关于遗传多样性的研究一直是R. solani研究的热点.本文就用于R. solani遗传多样性研究的方法进行了综述.分别解释并阐述了各种方法的优缺点，其中菌丝融合法是研究R. solani遗传多样性的传统方法，该法需借助显微镜且耗时耗力；同工酶法简便、高效、低廉，但常需要与其它方法联用；脂肪酸法操作难度小，价格相对较低，但该法受菌株生长状况和脂肪酸脂化方法的限制；分子标记法方法众多，各有利弊，通过比较发现rDNA-ITS是研究R. solani遗传多样性比较合适的方法.本文还介绍了不同方法在R. solani遗传多样性研究中的具体应用.     

有效分离1kDa小肽的Tricine-SDS-PAGE方法

为了建立能有效分离 1kDa的特小分子肽的Tricine SDS PAGE方法 ,调整分离胶中聚丙烯酰胺凝胶的分子组成 ,使致密胶中丙烯酰胺和双丙烯酰胺的交联度为 5 %,并加入 36 5 %的尿素 ,用于分离小至 1kDa的小分子肽 ,并与常规的SDS PAGE和先前报道的Tricine SDS PAGE相比较。结果改进的致密胶可以有效分离分子量小至 1kDa的小肽 ,明显优于常规的SDS PAGE和现有的Tricine SDS PAGE方法。     

齐整小核菌胞外多糖的研究*

鉴定了一株产胞外多糖的齐整小核菌,经纸层析、旋光度和红外光谱研究表明。该菌产生的多糖是由葡萄糖按β-键形成的多聚糖,可耐受高温、高盐和较广范圈的pH,具良好的增粘性和剪切稀释性能。     

草坪禾草丝核菌的核相研究

2003～2005年从上海市、浙江省、山东省、河南省和陕西省草坪褐斑病的97份病株标本中,分离得到了73个丝核菌分离物,其寄主包括多年生黑麦草Lolium perenne、高羊茅Festuca arundinacea、草地早熟禾Poa pratensis、匍匐翦股颍Agrostis palustris、结缕草Zoysia japonica和狗牙根Cynodon dactylon。用载玻片培养法和DAPI染色法对各分离物菌丝进行了染核观察。结果73个分离菌株可区分为单核丝核菌、双核丝核菌和多核丝核菌三大类,可能属于多个物种。单核丝核菌在中国为首次报道,同时也是其侵染草坪禾草的首次报道。     

真菌硬葡聚糖的生产及在油田上的应用

真菌小核菌 (Sclerotiumspp )产生一种新型非离子型抗盐抗温生物多糖聚合物 硬葡聚糖。综述了该糖的生产 ,发酵条件对生产的影响 ,及其在油田上的应用。     

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.