利用花粉管通道法获得转雪花莲凝集素基因(sgna)小麦

利用适用于禾谷类作物的表达载体pGU4AGBar和pGBIU4AGBar,采用花粉管通道法,将人工合成的雪花莲凝集素基因sgna导入了优良冬小麦品系西农2208和西农132,经PCR和Southern blot鉴定,证明获得了20株导入了sgna基因的转基因植株,转化率约为0．28％—0．84％,并通过Western blot鉴定检测到了目的蛋白的表达。     

利用花粉管通道法获得转雪花莲凝集素基因（sgna）小麦

利用适用于禾谷类作物的表达载体pGU4AGBar和pGBIU4AGBar,采用花粉管通道法, 将人工合成的雪花莲凝集素基因sgna导入了优良冬小麦品系西农2208和西农132,经PCR 和Southern blot鉴定,证明获得了20株导入了sgna基因的转基因植株,转化率约为0.2 8%～0.84%,并通过Western blot鉴定检测到了目的蛋白的表达。     

SOEing 法构建EGFP真核表达载体及其在杜氏盐藻中的表达

通过重叠区扩增基因拼接法（Gene splicing by overlap extension,SOEing）构建含有杜氏盐藻（Dunaliella salina）硝酸盐还原酶（NR）基因5′-上游序列（Pnr）and 3′-端序列（Tnr）的EGFP真核表达载体,并将其转化杜氏盐藻。利用改进的SOEing法,将杜氏盐藻NR基因Pnr与报告基因EGFP cDNA融合,并与pEGM-7zf克隆载体连接,顺序将盐藻NR基因Tnr序列与融合片段相连,构建含Pnr-EGFP-Tnr表达盒的盐藻真核表达载体p7NET。电击法转化杜氏盐藻,在盐藻转化株中观察到了EGFP的瞬时表达。此研究为转基因杜氏盐藻研究和成功建立杜氏盐藻生物反应器奠定了实验基础。     

核基质结合区的位置对转基因表达的影响

为研究核基质结合区 (MAR）序列不同插入位置对转基因表达作用的影响,PCR扩增人β 珠蛋白MAR分别插入到含氯霉素乙酰转移酶（chloramphenicol acetyltransferase,CAT）报告基因真核表达载体pCATG表达盒两侧、5′端及3′端.酶切鉴定后,用阳离子聚合物转染CHO细胞,G418筛选出阳性细胞克隆,ELISA分析CAT基因的表达水平,半定量PCR分析CAT基因相对拷贝数.结果表明,表达盒两侧含MAR序列的载体能提高介导的转基因表达水平平均提高10.4倍,5′端含MAR序列的载体表达水平平均提高3.9倍,3′端含MAR序列的载体反而降低转基因表达水平.5′端含MAR序列的表达载体其转基因相对拷贝数高于其它两组载体的基因拷贝数,转基因表达量与基因拷贝数不成正比.     

含VHb基因和GFMcryIA基因的双价基因植物高效表达载体的构建

将人工合成的、密码子优化后的透明颤菌血红蛋白基因与人工合成的GFMcryIA基因构建成双价基因植物高效表达载体PGBI4ASVHBBt,vgbM基因表达盒中含2个增强子的35S启动子、Ω前导序列、Kozak序列、多联终止密码子及Nos终止子;Bt基因表达盒中,除含有以上提高转录和翻译的调控元件外,还包含有正确切割、加工序列、Poly(A)信号序列。利用根癌农杆菌介导转化烟草,获得了转基因植株;PCR及Southern blot检测,证实了双价基因在烟草基因组中的整合;Western blot检测证实了vgbM基因在转基因烟草中的表达;杀虫实验表明GFMcryIA基因也表达出活性毒蛋白。     

类球红菌自诱导物合成酶基因cerⅠ的克隆及其原核表达

根据类球红菌（Rhodobacter sphaeroides2.4.1）自诱导物合成酶基因cerⅠ的序列,设计并合成了1对特异性引物,在引物的5′和3′分别加入含有HindⅢ和XhoⅠ限制性酶切位点的序列,以类球红细菌Rhodobactersphaeroides基因组为模板扩增了cerⅠ基因序列。将PCR产物与pMD18-T载体连接,转化大肠杆菌DH5α。鉴定成功获得目的片段,经HindⅢ和XhoⅠ双酶切后与载体pET-28a（＋）连接,构建原核表达质粒pET-28a（＋）-cerⅠ,并将其转化宿主菌BL21（DE3）,用IPTG诱导其表达。SDS-PAGE分析表明,重组载体pET-28a（＋）-cerⅠ可成功地在大肠杆菌中表达cerⅠ蛋白。     

含VHb基因和GFMcryIA基因的双价基因植物高效表达载体的构建

将人工合成的、密码子优化后的透明颤菌血红蛋白基因与人工合成的GFMcryIA基因构建成双价基因植物高效表达载体PGBI4ASVHBBt,vgbM基因表达盒中含2个增强子的35S启动子、Ω前导序列、Kozak序列、多联终止密码子及Nos终止子;Bt基因表达盒中,除含有以上提高转录和翻译的调控元件外,还包含有正确切割、加工序列、Poly(A)信号序列。利用根癌农杆菌介导转化烟草,获得了转基因植株;PCR及Southern blot检测,证实了双价基因在烟草基因组中的整合;Western blot检测证实了vgbM基因在转基因烟草中的表达;杀虫实验表明GFMcryIA基因也表达出活性毒蛋白。     

cmv-3′LTR嵌合启动子对逆转录病毒载体MFG滴度及外源基因表达的影响

为减轻逆转录病毒载体介导的外源基因的沉默,进一步提高逆转录病毒载体MFG介导的外源基因的表达水平,同时探讨逆转录病毒3′端长末端重复序列(long terminalrepeat,LTR)内U3区对病毒基因表达的影响,将逆转录病毒载体3′端LTR内的U3区用cmv核心增强子、启动子序列替代,同时去除了3个与逆转录病毒载体启动子甲基化有关的序列NCR、DR,并以egfp为报告基因,构建了MFG egfp和MFG egfp cmv表达载体。结果显示:利用cmv启动子替代MFG载体3′端LTR的U3启动子序列,会显著降低MFG载体的病毒滴度及其介导的外源报告基因的表达。提示利用cmv启动子替代病毒载体的3′端LTR内的U3区,并不是提高MoMLV(moloneymurineleukemivirus)逆转录病毒载体介导外源基因的表达及其病毒滴度的理想策略。结果也同时提示:在MoMLV逆转录病毒3′端LTR的U3区,可能存在与病毒RNA加工、成熟及稳定性有关的信号序列。     

香蕉果实成熟相关基因ACO1启动子区的克隆及其功能初探

根据已报道的香蕉课实表达ACC氧化酶基因（ACO1）的序列,用改进的接头连接PCR法从香蕉基因组中扩增并克隆了此基因5′旁侧区1526bp的片段,其中包含一个推测的TATA盒序列;与已公布的两个香蕉ACC氧化酶基因启动子序列（分别为934bp和1451bp）的相似性各为97．3％（Lopez-Gomez等）和88．8％（May和Kipp)。将4个含有不同大小启动子区的克隆片段与GUS基因编码区连接构建成嵌合成基因,通过基因枪轰击转入香蕉叶、根和果实的细胞后,瞬时表达结果表明不同大小的ACO1启动子区段都只在果实细胞中指导GUS基因表达,证明该启动子具有指导基因在果实中表达的功能,并推测负责果实特异性的顺式元件可能位于启动子近端0．7kb区段之内,在468至822的355bp区段内可能在与正控制有关的顺式元件。     

香蕉果实特异性ACC合酶基因启动子区的克隆及其功能初探

根据本实验室所获得的香蕉果实特异性ACC合酶cDNA序列,以改进的接头连接PCR方法通过两次步行从香蕉基因组中分别扩增并克隆了其基因5′旁侧区近端1.2kb和远端1.6kb的片段。通过拼接,构建出含有2505bp启动子区和转录起始位点下游86bp的共2591bp的基因5′旁侧区片段;其启发性动子区中34至28为推测的TATA盒序列,158至146为推测的CCAAT盒,与其它植物基因启动子结构相类似。将2.5kb启动子片段与β-葡糖苷酸酶(GUS)基因编码序列融合,用基因枪法将构建的嵌合基因转入香蕉叶、根和果实的细胞后,只在果实细胞中观察到报告基因的瞬时表达,从功能上证明了此25kb的启动子片段具有指导报告基因在香蕉果实中特异性表达的作用。同时构建5个含不同5′端缺失启动子与GUS融合基因的表达载体。瞬时表达结果表明可能负责果实特异性表达的调控区存在于转录起始位点至-1111的启动子区中,而在-1111至-608区间可能存在一个正控制区。     

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.