胃癌相关cDNA片段的快速克隆和表达分析

利用差异显示PCR技术获得的一条在胃癌和正常组织有差异表达的表达序列标签(EST)-W123(GenBank登录号为AF150631),通过与GenBank的dbest库进行电子杂交,选取了与其同源度高的若干EST,在它们共有的保守序列设计了用于扩增的寡聚核苷酸引物,利用cDNA末端快速扩增PCR（RACE）技术得到了7条带有polyA尾的3′EST,进行序列分析后,发现它们均是代表新基因或不同剪接体的EST,且具有共同的保守序列,已登录GenBank.采用RNA印迹对目的序列进行初步鉴定,并进行了这些基因的组织分布分析.RACE技术和生物信息学相结合,具有快速、高效的特点,有助于疾病相关基因的克隆.     

小麦NBS-LRR类抗病基因同源cDNA序列的分离与鉴定

利用抗病基因的保守结构设计引物,从抗叶锈病近等基因系材料TcLr24中扩增出一条703bp的条带RGAl,通过与GenBank比对,选取与RGAI高度同源的若干条带,在它们共有的保守序列位置设计引物,利用cDNA末端快速扩增(RACE):ffL术扩增抗病同源基因cDNA全长.扩增到3条全长cDNA,经BLASTp比较,这些序列都舍有NBS保守结构域和多个LRR结构域.与很多已知植物抗病基因的功能相应区域一致.对FRGA-1,、FRGA-2和FRGA-3实时定量PCR分析,表明这3个基因在小麦叶片中都是组成型表达.本研究在小麦材料TcLr24中得到3条抗病基因同源cDNA全长,为研究小麦抗病基因奠定了基础.     

梅EST-SSR标记的开发及利用

利用MISA软件对10 123条梅EST序列进行SSR位点查找,得到含SSR位点的序列935条,SSR位点1233个,平均每100条EST序列中含有12.18个SSR位点.2核苷酸、3核苷酸重复是最主要的重复类型,分别占35.52%和41.36%.设计了40对EST-SSR引物并进行扩增,有24对引物能扩增出理想的PCR 产物,其中17对引物具有较好的扩增多态性.测序后发现13对引物中有73.08%的片段具有相应的SSR位点,对杏DNA指纹中部分谱带的测序结果也证明了是梅扩增出的相应的SSR位点.根据本研究含有SSR位点的测序结果推算,从梅EST中开发真实SSR位点的数目为901.随机选择13对引物对杏和梅进行DNA指纹构建与遗传多样性分析,结果发现,来源于梅的EST-SSR引物在杏中有很高的通用性,这些引物把梅和杏分成了两大群体,说明他们是遗传差异明显的两种植物.     

一种新的cDNA末端快速扩增获取全长cDNA的方法

为克隆精子发生相关基因的全长cDNA,根据mRNA差异显示获得的ESTs设计引物。利用一种新的cDNA末端快速扩增方法（SMART RACE）扩增该EST的5′末端,并进行克隆测序,与cDNA差异显示获得ESTs拼接后,获得了三个新的全长cDNA。结果表明：SMAR RACE是一种简便、有效的克隆cDNA5′末端未知序列的技术。     

水花生Actin基因片段的克隆及序列分析

肌动蛋白基因编码生物体内一种很重要的组成型表达蛋白质,常被看作看家基因。为分离水花生肌动蛋白基因,根据GenBank上一藜科植物的———Actin基因的EST序列,分别设计引物,提取水花生根的总RNA,对上述的基因片断进行RT—PCR,扩增后测序得到230bp长的核苷酸序列。经同源性比较,这条序列与上述藜科植物Actin的EST序列基本一致,表明获得了水花生肌动蛋白序列。     

小麦抗赤霉病品种NBS同源序列克隆与分析

根据二穗短柄草NBS-LRR类基因的保守序列设计同源引物,以小麦抗赤霉病品种苏麦3号、宁7840和望水白基因组DNA为模板,通过PCR扩增,得到43条序列,其中4条为非编码序列或结构域不完整;39条与植物抗病基因同源,其中的7条内部存在终止密码子,可能是假基因,经过比对分析,其余32条具有连续的开放阅读框和保守结构域,推导的氨基酸序列均具有Kinase-1a、Kinase-2和Kinase-3a及GLPL区等几个保守区,在GenBank中均能找到与之高度同源的其他物种的核酸序列,并且Kinase-2的最后一个氨基酸均为色氨酸(W),属于non-TIR类NBS基因。32条序列可分为4大类,它们之间核苷酸同源性为64%-98%,编码氨基酸同源性为22%-98%。根据序列分析随机设计5对不同基因特异性引物,并利用RT-PCR技术进行表达分析,结果表明,7-1、s-3、s-4和w-2均能表达,说明这些片段可能是功能性抗病基因的部分序列;7-13不表达,再次证明属于假基因。32条序列在之前未被报道过,这些RGA可以作为筛选赤霉病功能性抗病基因的候选序列。     

白桦雌花序抑制性消减文库构建及EST分析

为研究白桦雌花序发育过程中特异基因的表达,以白桦雌花序样品为tester,雄花序样品为driver,利用SMART策略构建了白桦雌花序抑制性消减（SSH）文库。构建SSH文库的重组率为72%,插入片段的平均长度为400 bp左右。随机挑选文库克隆测序,获得150条EST序列,这些序列被GenBank的dbEST数据库收录,收录号为EE284580-EE284681,EE595316-EE595363。通过BlastX对EST进行功能注释,并对其中同源性较高的111条EST按功能进行分类,EST功能涉及了代谢、细胞防御、转录调节、能量代谢及信号传导等途径。发现了多个已知的控制花发育相关的EST,它们占已知功能EST的21%其功能涉及到调控花序形成和花分化、调控花粉与柱头亲和性以及调控花粉管发育等,包括MADS-box、S-locus F-box等基因。这些EST的获得为了解白桦花期基因表达,白桦花发育相关基因克隆和功能解析奠定了基础。     

漆树EST-SSR引物开发

利用NCBI数据库进行漆树EST-SSR引物开发,从NCBI数据库中共下载漆树EST序列87 856条。利用MISA软件进行序列处理、拼接及聚类后,从87 856条漆树EST序列中拼接组装成3 979条非冗余序列,含SSR位点的EST序列出现频率占EST序列总数的4.5%,从3 979条非冗余序列中检测到487个SSRs微卫星位点,出现频率为12.2%。这些SSR位点中,三核苷酸和二核苷酸重复基元所占比例较高。采用Primer5.0软件,共成功设计50对EST-SSR引物,50对EST-SSR引物在25个漆树个体上均能扩增出清晰的电泳条带,其中18对引物检测出了多态性条带,扩增率达36%。     

中华鲟铁蛋白基因cDNA全长克隆与组织表达分析

根据铁蛋白基因的保守序列,搜索GenBank数据库中华鲟的EST数据库得到一条同源序列.通过RT-PCR的方法对该序列进行扩增,修改其测序错误,获得中华鲟铁蛋白亚基cDNA全长,经过注释提交GenBank数据库,获取序列登录号EU348782.该cDNA长度为896bp,包含531bp的完整编码区,推测编码的蛋白质为176aa,分子量为20339.9Mr,理论等电点为5.66.它和大两洋鲑鱼铁蛋白序列同源性最高,达到82.9%.该基闪在中华鲟肝脏、胰脏、肌肉、脑、心脏、鳃和胃粘膜等多种组织表达,在胰脏和心脏中表达量较高,在肌肉组织中表达较低.根据同源模建的方法得到该蛋白质三维结构,其包括5个α螺旋和10个转角结构,和人、蛙和细菌的铁蛋白均能很好的叠合,表现了很高的相似性,表明该蛋白结构和功能在基因进化中的高度保守性.     

中华鲟铁蛋白基因cDNA全长克隆与组织表达分析(英文）

根据铁蛋白基因的保守序列,搜索GenBank数据库中华鲟的EST数据库得到一条同源序列。通过RT-PCR的方法对该序列进行扩增,修改其测序错误,获得中华鲟铁蛋白亚基cDNA全长,经过注释提交GenBank数据库,获取序列登录号EU348782。该cDNA长度为896 bp,包含531bp的完整编码区,推测编码的蛋白质为176 aa,分子量为20339.9 Mr,理论等电点为5.66。它和大西洋鲑鱼铁蛋白序列同源性最高,达到82.9%。该基因在中华鲟肝脏、胰脏、肌肉、脑、心脏、鳃和胃粘膜等多种组织表达,在胰脏和心脏中表达量较高,在肌肉组织中表达较低。根据同源模建的方法得到该蛋白质三维结构,其包括5个α螺旋和10个转角结构,和人、蛙和细菌的铁蛋白均能很好的叠合,表现了很高的相似性,表明该蛋白结构和功能在基因进化中的高度保守性。     

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.