通过DNA改组技术获得高活性β-葡萄糖苷酸酶

β 葡萄糖苷酸酶是在植物转基因中广泛应用的报告基因 .以质粒pBI12 1中的GUS基因为基础 ,利用DNA改组方法 ,经DNaseⅠ降解 ,PrimerlessPCR ,PrimerPCR对GUS基因进行了突变和改组 ,然后将改组的GUS基因连接到原核表达载体pG2 5 1中 ,构建了库容为 10 8的突变体库 .经过活性的筛选 ,得到活性提高的克隆 ,再以此为基础 ,经过新的改组、筛选得到活性大幅度提高的克隆GUS2 4 .基因测序显示 ,GUS2 4与GUS基因之间的同源性为 99 7% ,共有 6个核苷酸位点发生了改变 ,分别是 :379位的A突变为G ,396位的T突变为C ,711位的G突变为A ,95 8位T突变为C ,990位的T突变为C ,1649位的A突变为G .核苷酸序列推导的氨基酸序列显示 ,3个氨基酸发生了突变 ,12 7位的Ser突变为Gly ,32 0位的Trp突变为Arg ,5 5 0位的Asn突变为Ser.X gluc染色检测和荧光测活结果显示GUS2 4基因表达的 β 葡萄糖苷酸酶基较GUS基因表达产物活性提高 3倍     

7-木糖紫杉烷糖基水解酶Lxyl—pl-2基因突变库与高通量筛选体系的构建

对7－木糖紫杉烷糖基水解酶Lxyl－p1－2基因进行定向进化的初步研究，确定易错PCR条件、优化克隆连接方法、建立基于48微孔板高通量筛选模型。利用易错PCR技术对Lxyl－pl－2基因进行随机突变，比较不同浓度M聋’进行易错PCR，每组随机挑取10个克隆进行测序。序列分析表明：在M聋’浓度为7mmol／L条件下，碱基平均突变率为0．12％，即对于Lxyl－p1－2基因（2．4kb）来说，每个基因序列平均有3个碱基突变，达到构建突变文库的频率要求，选择该条件作为易错PCR条件。利用in-fusion技术将Lxyl－p1－2突变基因克隆到pPIC3．5K载体中，获得大量重组突变质粒。该研究优化了in-fusion连接反应中基因片段和载体片段的摩尔比，并探讨了基因片段与载体片段问同源序列长度对in—fusion连接效率的影响。试验结果表明：基因片段与载体片段的摩尔比最佳梯度为5：1；同源序列长度的选择成为影响in—fusion连接效率的关键因素之一。当同源序列长度为100bp时连接效率达到最大值，且显著高于同源序列长度为15～50bp的连接效率，此时阳性重组率提高至90％左右。与常规酶切一连接法相比，in-fusion法具有明显优势，同时将克隆周期由3～4d缩短为1～2d。挑取单菌落，在48微孔培养板上进行培养、诱导表达2d后，取菌液进行酶活测定（底物PNP-Xyl）。以野生型为例，β-木糖苷酶的酶活013405均数μ=3．415，其数据组标准差为δ=±0．078，数值符合正态分布的原理，建立统计学野生型均数分布范围和48微孔板高通量筛选，为后续突变体筛选提供基础。     

从人血液中克隆过氧化氢酶基因

目的从人血液中克隆过氧化氢酶基因。方法以新鲜的人血液为材料,提取全血RNA,运用RT—PCR方法扩增过氧化氢酶基因,构建pMDl8T／CAT克隆载体,并进行了不同来源过氧化氢酶的氨基酸序列同源分析。结果从人血液中成功扩增出过氧化氢酶基因,获得了重组载体PMD-18T／CAT,氨基酸序列分析的同源性达80％以上。结论从人血液中可以很方便地克隆出过氧化氢酶基因。     

2,5-DKG还原酶Ⅱ基因的克隆和在大肠杆菌中的表达

参照文献上的2,5-二酮基-D-葡萄糖酸（简称2,5-DKG）还原酶II基因序列,合成两个引物序列并在两端加上EcoRI和BamHI两个酶切位点,抽提棒状杆菌SCB3058菌株的染色体为模板进行PCR反应,克隆得到2,5-DKG还原酶II基因,酶切验证与预期的结果相符合。将此片段克隆到pGEM-T载体上保存．将2,5-DKG还原酶II基因用EcoRI和BamHI内切酶切下,连接到pBV220载体上,构建成表达载体。42℃诱导不能得到稳定的蛋白表达条带和酶活力,测序发现基因的3’末端的原PCR引物外少合了一个碱基,终止密码子发生移码突变而消失。此外在5’端的启始密码子ATG前有三个碱基与pBV220载体上的SD序列发生配对。据此,重新设计和合成了PCR引物,并用pBV220和pBL4载体构建了两个表达载体。42℃诱导表达均得到了稳定的表达条带和较高的酶活力．     

拟南芥生长素受体基因TIRl启动子的克隆及序列分析

以野生型拟南芥（Arabidopsis Columbia）基因组DNA为模板,通过PCR扩增得到拟南芥生长素受体基因TIRI启动子2008片段,将该片断克隆到PGM-T载体上。序列分析表明,该启动子大小为2008bp,RNA聚合酶识别序列TATA-box,TIR1特异表达和增强序列CAAT-box皆完整,与已报道的序列比较仅有3个核苷酸发生改变,同源性为99．85％。将该启动子与GUS基因融合,构建成表达载体后,在拟南芥和烟草叶片中做瞬时表达,结果分析显示：拟南芥和烟草叶片中均有GUS酶活性存在。     

小鼠白细胞介素-33基因的克隆及序列分析

目的:克隆小鼠IL-33基因全长编码区cDNA,并对其进行序列分析。方法:从BALB/c小鼠的脊髓组织中提取总RNA,逆转录为cDNA,用热启动PCR技术,扩增小鼠IL-33基因全长编码区cDNA,经双酶切后,克隆入pcDNA3.1( )载体中,构建真核表达载体pcDNA3.1-mIL-33,然后进行酶切鉴定与序列分析。结果:小鼠IL-33基因的PCR产物和重组载体经凝胶电泳和酶切鉴定、测序分析证实,其序列与GenBank中数据一致。小鼠IL-33基因的全长编码序列为801 bp,编码266个氨基酸。结论:小鼠IL-33基因成功的克隆并构建了其真核表达载体,为进一步进行IL-33的表达与功能研究奠定了基础。     

bax转录调控pGL3-Basic荧光素酶报告基因载体的构建

目的:构建能用来检测bax转录调控作用的报导基因载体.方法:采用PCR方法克隆出bax 5'UTD基因,构建载体后利用酶切与测序进行鉴定.然后将该基因亚克隆至pGL3-Basic报告基因载体上.结果:克隆基因产物酶切结果与理论预测值一致,测序未出现一个碱基突变.PCR鉴定发现,bax5'UTD正确连接到了pGL3-Basic报告基因载体上,构建了pGL3-Basic-Baxregular载体.结论:bax 5'UTD报导基因载体的构建为进一步研究bax转录调控作用提供了载体资源.     

紫杉烷13α-羟基化酶基因的克隆及其转化烟草的研究

本研究利用东北红豆杉(Taxus cuspidata )cDNA文库作为模版,通过PCR技术扩增得到我国东北红豆杉紫杉烷13α-羟基化酶(Taxane 13α-hydroxylase,简称13OH)的全长cDNA,将PCR产物克隆到pGM-T载体后测序结果表明该序列长度为1458bp。同源性比较分析结果表明：其碱基序列与已经报道的东北红豆杉(Taxus cuspidata)的13OH基因的一致性为99.38%,其氨基酸序列同与已经报道的东北红豆杉的13OH氨基酸序列的一致性为99.18%。将获得的cDNA全长序列正向插入到含有GUS报告基因的pCambia1305.1后成功地构建出东北红豆杉13OH植物表达载体pC13OH,通过电击法把pC13OH转入根癌农杆菌GV3101中。利用该工程菌株对普通烟草进行了转化,在潮霉素选择压力下获得了完整的再生植株。利用13OH基因特异引物,通过PCR技术筛选到4株阳性再生植株。在这4株再生植株中,有3株植株GUS报告基因的组织化学染色呈现阳性反应,表明该植物载体表达载体中与13OH相融合的GUS基因成功地得到了表达。本研究为今后深入研究13OH基因在烟草中的表达和开展紫杉烷13α-羟基化酶基因对红豆杉细胞的转化以及研究作用于该基因的小RNA调节子打下了基础。     

FLT-1启动子荧光素酶报告基因重组体的构建和鉴定

目的：为探索FLT-1启动子靶向调控活性分析,克隆FLT-1启动子基因序列,构建并鉴定肿-1启动子调控的荧光素酶报告基因重组体pGL3-FLT—Basic—luc。方法：应用聚合酶链式反应（PCR）技术扩增FLT-1启动子序列,定向亚克隆至荧光素酶表达载体pGL3-Basic—luc中,构建含有正确目的基因的报告基因重组体pGL3-FIT—Basic—luc,通过限性内切酶酶切、PCR及测序进行鉴定。结果：通过酶切鉴定及基因测定证明,所克隆的基因产物与预期结果-致,序列无碱基突变。结论：成功构建了含有FLT-1启动子基因序列的荧光素酶报告基因真核表达载体,为下-步分析该启动子活性及血管疾病的基因治疗奠定基础。     

小鼠Daintain/AIF-1 基因启动子的克隆及其荧光素酶报告基因 载体的构建

目的:对Daintain/AIF-1(大炎肽/同种异体移植炎症因子-1)基因启动子进行克隆并构建荧光素酶报告基因载体,为进一步研究Daintain/AIF-1的转录调控作用提供了质粒资源。方法:提取单核巨噬细胞系RAW264.7基因组DNA,以其为模板采用PCR方法克隆出Daintain/AIF-1基因5’端UTR区1.6 kb DNA序列,将该序列同源重组到pGL3-Basic载体上,转化感受态DH5α并酶切鉴定和测序。结果:PCR产物片段与预期结果一致,Daintain/AIF-1基因5’端UTR区1.6 kb DNA序列连接到pGL3-Basic载体上,构建成pGL3-Basic-Daintain/AIF-1(pGL3-Basic-DT)载体,酶切结果与理论预测值一致,经测序证实无碱基突变。结论:Daintain/AIF-1基因报告基因载体的构建为进一步研究Daintain/AIF-1转录调控作用提供了载体资源。     

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.