phbB,phbC基因克隆,序列分析及植物表达载体的构建

利用聚合酶链式反应技术,从真养产碱杆菌Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｈｕｓ Ｈ１６染色体ＤＮＡ中的主增并克隆了调控聚－β－羧基丁酸生物合成的两个关键酶基因;依赖ＮＡＤＰＨ的乙酰乙酰ＣｏＡ还原酶基因和ＰＨＢ合成酶基因。限制性内切酶图谱和核苷酸序列分析证实了克隆结果,并表明所克隆的基因与国外报道的有很高的同源性。     

人粒细胞集落刺激因子cDNA的克隆、序列鉴定及初步表达

利用多聚酶链反应技术,从人膀胱癌细胞系5637细胞中快速扩增并克隆了人粒细胞集落刺激因子cDNA,序列分析证明,该cDNA包含人粒细胞集落刺激因子的全部编码基因,全长612bp,编码30个氨基酸的信号肽和174个氨基酸的成熟蛋白。其中第43位codon出现一个碱基的突变(CAC→TAC)导至第43位氨基酸的改变(组氨酸→酪氨酸)。经逆转录病毒导入SP2/0细胞并初步表达。结果表明:该基因产物具有G-CSF活性。     

cIts857基因的克隆、修饰及温敏诱导表达载体

从噬菌体λＤＮＡ（ｃＩｉｎｄｌｔｓ８５７Ｓａｍ７）克隆出９９０ｂＰ的ｃＩｔｓ８５７基因,经缺失,点突变等修饰改造和ＤＮＡ序列测定,获得了带有自身启动子区的修饰型ｃＩｔｓ８５７基因（７７０ｂｐ）。进而利用它构建大肠杆菌表达载体ｐＢＬＭＶＬ２和一套含３种不同阅读框架ｌｉｎｋｅｒ区的表达载体ｐＢＬＭＦＶ４、５Ｂ、６。上述表达载体;用于表达人工合成的牛生长激素（ｂＧＨ）、人干扰素－γΔＣ－１３和酵母ｐｈｏ８５等外源基因,都观察到这些基因产物的温敏表达。这些表明,克隆、修饰并组建到表达载体中的λ噬菌体ｃＩｔｓ８５７基因表达出具有功能作用的转录阻遏蛋白,使上述ＰＬ启动子控制下的大肠杆菌表达载体可经温敏诱导而高效表达外源基因产物．     

拟南芥AtVQ29基因的克隆与植物表达载体构建

目的:VQ模序蛋白是植物中所特有的一类具有高度保守序列的蛋白质,广泛参与植物的生长发育与逆境反应,本研究拟克隆拟南芥的AtVQ29基因并进一步构建由组成型启动子CaMV 35S驱动的植物表达载体pSN1301-AtVQ29。方法:采用CTAB法提取拟南芥基因组DNA,根据已报道的AtVQ29基因序列设计并合成引物,通过PCR技术扩增获得拟南芥AtVQ29基因,经T载体克隆后测序。利用生物信息学软件对序列进行初步分析,同时基于基因重组技术构建植物表达载体。结果:序列分析表明已成功克隆AtVQ29基因,该基因编码区全长为372bp,共编码123个氨基酸残基,具有保守的VQ模序。并进一步构建了由组成型启动子CaMV 35S驱动的AtVQ29基因植物表达载体pSN1301-AtVQ29。结论:本研究所构建的AtVQ29基因植物表达载体能够在转基因植株中过量表达AtVQ29基因,为后期开展基因功能研究与植物基因改良奠定了基础。     

Cloning and Sequencing of the Gene Encoding 10 kD Zein of Maize

The results of cloning and sequencing of the gene encoding 10 kD zein of maize (Zea mays L. ) with polymerase chain reaction (PCR) technique are here with presented. The genomic DNA template was extracted from sterilized seedlings of maize. Primered with a pair of synthetic 5′and 3′ PCR primers, a 0.57 kb DNA fragment was obtained after 30 PCR amplification cycles. The restriction map of the DNA fragment has been determined. The result indicated that the entire coding sequence of 10 kD zein gene has been cloned. The homologies of the DNA sequence and deduced amino acid sequences between our result and those published abroad are 96% and 90% respectively. As l0 kD zein is rich in sulphur, this gene might be used to improve the quality of crops, especially, the forage legumes by genetic engineering.     

Construction of Plant Expression Vectors and Identification of Transgenic Potato Plants

Potato virus X (PVX), potato virus Y (PVY) and potato leaf roll virus (PLRV) infection in potato may result in the loss of centrification of seed potatoes and affect the quality and yield of potatoes in agricultural production. The authors cloned coat protein (cp) genes of PVX, PVY and PLRV and constructed two kinds of plant expression vector which contain PVX and PVY or PVY and PLRV cp genes. Three major commercial cultivars of potato and one cultivar of tobacco were transformed via Agrobacterium tumefaciens mediated procedure. Transgenic plants were confirmed by PCR analysis. Transgenic tobacco plants containing both PVX and PVY cp genes were significantly resistant to PVX and PVY infection via mechanical inoculation.     

用于通路(Gateway)克隆技术的植物表达载体研究进展

通路(Gateway)克隆技术是根据λ噬菌体基因组和大肠杆菌基因组之间的位点专一性重组分子机制开发的一套分子克隆新技术.利用该技术LR反应构建目的基因的表达载体时不需要经过酶切和连接等繁琐而又费时的过程,因此,可以节省很多时间.为了扩大Gateway技术在植物基因工程领域的应用,最近有很多研究机构和研究小组开发了能用于组成型或诱导型表达目的基因、基因沉默、启动子分析、蛋白质亚细胞定位、蛋白质/蛋白质相互作用、多个DNA片段的模块化组装和DNA组片段功能验证等研究用的植物表达载体.该文对这些技术的研究进展进行了综述.     

选择标记可去除的植物高效表达载体的构建

在常用的植物组成型表达载体pBI121的选择标记基因NPTII两侧插入同向的lox位点并用多克隆位点(MCS)取代了GUS基因序列,构建了NPTII基因可被去除的和可插入目的基因的通用植物表达载体pBI121-lox-MCS。替换pBI121-lox-MCS中驱动目的基因表达的35S启动子,可构建成一系列具有其他表达特性的植物表达载体,如本文描述的韧皮部特异表达载体pBdENP-lox-MCS。为方便地筛选去除选择标记基因的转基因植物,还构建了绿色荧光蛋白(GFP)表达框与NPTII表达框连锁的pBI121-gfp-lox-MCS载体。上述植物表达载体可广泛应用于培育选择标记可去除的转基因植物。     

小麦淀粉合酶基因III片段的克隆及反义和RNA干扰载体的构建

采用RT-PCR方法从小麦品种豫教2号的发育籽粒中克隆出淀粉合酶III基因（starch synthase III, SSIII）部分cDNA片段(509bp) (GenBank No. EF466009),同源性比较结果显示,它与GenBank 上已报道的SSIII基因有高度同源性。以pWM101质粒为基础,构建了由35S启动子调控的SSIII基因的反义表达载体pWM101SSIII;另外,还以pFGC5941质粒为基础,构建了SSIII基因的RNAi干扰载体pFGC5941SSIII,这些载体的构建为研究此基因的功能打下了很好的基础。     

用PCR突变技术克隆艾滋病病毒蛋白酶基因

作者设计并合成了一对用于PCR技术的突变引物HIV-1 Pr1和HIV-1Pr2,分别在两引物中设计了两个突变点,使突变后基因含有EcoRI、HindⅢ和TAA序列,便于HIV-1 Pr基因的定向克隆和表达。用HIV-1 Pr1和HIV-1 Pr2作引物,采用PCR方法从HIV-1基因组DNA中扩增出了一个360bp长的DNA片段,用EcoRI和HindⅢ双酶切法将此片段定向克隆入pUC19质粒,将克隆基因插入M13mp18进行DNA序列分析。结果表明,该基因序列的读框完全正确,从而为HIV-1 Pr基因的表达及抑制剂的研究奠定了基础。     

小麦HMW-GS 12基因克隆及植物表达载体构建

目的:为了利用基因遗传转化改良小麦品质,采用聚合酶链式反应(PCR)技术。方法:从小麦品种东农7742基因组DNA中扩增并克隆了小麦高分子量谷蛋白12亚基基因(HMW-GS 12)。结果:序列分析结果表明,该基因全长1 980bp,其核苷酸顺序和推导的氨基酸顺序与已发表的序列相比,同源性分别为99.5%和99.7%。经过基因拼接,分别构建了胚乳特异性表达和组成型表达的高分子量谷蛋白12亚基基因的两个植物表达载体pDNPPBIHG和pUbPBIHG。     

以木糖异构酶基因xylA为选择标记的Gateway系统植物表达载体的构建

目的:构建以木糖异构酶基因xylA为筛选标记的无抗生素标记Gateway系统植物表达载体。方法:克隆大肠杆菌木糖异构酶基因xylA并用其替换植物表达载体pCAMBIA1301中的hpt基因,利用载体中的多克隆位点将Gateway Binary Vector(pH7WG2D)中酶切位点XbaⅠ和HindⅢ之间包括P35S、T35S、attR1、attR2和CmR-ccdB的片段重组入表达载体pCAMBIA1301中,构建表达载体pCAMBIA1301-xylA-GW,利用含有津田芜菁HY5基因片段的BP反应产物与载体进行LR反应,获得含有目的基因的植物表达载体pCAMBIA1301-xylA-HY5,并导入根癌农杆菌LBA4404中。结果:抗生素筛选及酶切和PCR鉴定表明成功构建了以xylA为筛选标记的无抗生素标记植物表达载体pCAMBIA1301-xylA-HY5。结论:利用木糖异构酶基因xylA结合Gateway克隆技术构建无抗生素标记植物表达载体,可简化、方便植物转基因表达载体构建。     

The cDNA Cloning and Nucleotide Sequence of the Gene Encoding Nonstrucure Protein of Rice Dwarf Virus Genome Segment 10

The results of cloning and sequencing the gene encoding nonstructure protein of the rice dwarf virus (RDV) gtnome segment 10 with polymerase chain reaction(PCR) technique were reported. The amplified PGR product was cloned into Hind Ⅱ site of plasmid pGEM3zf(-) and analysed with restriction enzymes. The physical map of the cloned fragment has been constructed, the insert is 1150 bp in length with restriction enzyme sites of Sac Ⅰ, Hind Ⅲ, NdeⅠ, BamH Ⅰ, etc. Besides, two restriction enzyme sites Bgl Ⅱ and EcoR Ⅰ have been separetely added in the 5 and 3 end of the segment in order to be cloned into plant intermediate vector in a convenient way. The fragments cleaved by the above-mentioned restriction enzymes were subcloned and the DNA sequence of full length of segment 10 was determined. In comparison with the RDV epidemic in Japan, the nucleotide sequence and deduced amino acid sequence of cloned segment 10 are 96.03% and 97.17% in homology respectively.     

抗人黑色素瘤单链抗体基因的克隆和分泌型表达

应用ＲＴ－ＰＣＲ技术从分泌抗人黑色素瘤单克隆抗体的杂交瘤细胞ＨＢ８７６０中克隆了抗体轻、重链可变区基因,然后用（Ｇｌｙ４Ｓｅｒ）３连接肽基因将ＶＨ、ＶＬ连接成ＳｃＦｖ基因,并进行了序列测定．计算机分析表明ＶＨ,ＶＬ均符合小鼠抗体可变区的特征,为功能性重排的抗体可变区基因．ＶＨ、ＶＬ、ｌｉｎｋｅｒ拼接正确．ＳｃＦｖ基因全长７２９ｂｐ,其中ＶＨ基因长３６０ｂｐ,编码１２０个氨基酸,ＶＬ基因长３２４ｂｐ,编码１０８个氨基酸．在噬菌粒表达载体ｐＣＡＮＴＡＢ５Ｅ中表达了可溶性的ＳｃＦｖ蛋白,表达产物经流式细胞仪检测可特异地与黑色素瘤细胞结合,不与肝癌、胃癌及良性黑痣细胞结合     

10kD玉米醇溶蛋白基因的克隆与植物表达载体构建

由于紫花苜蓿中的含硫氨基酸水平很低,采用基因工程手段将富硫蛋白基因-10kD玉米醇溶蛋白(zein)基因转入苜蓿中以提高其水平。根据该基因的保守序列设计引物,并在下游引物终止密码子前引入内质网驻留蛋白信号序列(KDEL),通过PCR技术从玉米(Zea mays L.)黄化幼苗基因组中扩增目的基因的开放阅读框(ORF)。序列测序得到全长为465bp的目的片断,该片段编码154个氨基酸,其中甲硫氨酸(M)31个占20.13%,半胱氨酸(C)6个占3.90%,含硫氨基酸共占24.03%。该基因与报道的10kD玉米醇溶蛋基因具有很高的同源性。将该基因构建到植物表达载体pCAMBIA13011上,以转化紫花苜蓿提高其含硫氨基酸的水平。     

phbB,phbC Gene Expression in E. coli and Their Transformation and Identification in Potato

Using NADPH-dependent acetoacetyl-CoA reductase gene (phbB) and poly-β-hydroxybutyrate (PHB) synthase gene (phbC) cloned from Alcaligenes eutrophus H16 and expression vector pKK223-3, the authors constructed an E. coli expression vector pKCB containing independent phbB and phbC operators, respectively, and transfered it into E. coli JM109. The microscopy and GC analysis indicated that E. coli JM109 (containing pKCB) induced by IPTG could synthesize poly-β- hydroxybutyrate (PHB). By DNA processing, three tuber-specific plant expression vectors, pP- SAGB (containing phbB), pBIBGC ( containing phbC) and pPSAGCB ( containing both phbB and phbC), were successfully constructed. In 5 transformed potato cuhivars, the authors screened 20 positive lines.     

商陆抗病毒蛋白cDNA的克隆,测序及其植物表达载体的构建

商陆抗病毒重【'1（m）卜。。）（1。Ill'l－。l。。111。。。""。',NP）是从美洲商陆叶片。I。分或Utoot件如VR,分子量约30hi）L'。PAP属典型的中链核栩然大活主l'I,对动物病毒和植物病;每均计）'们的抗病;沙作用','－。在国外,PAP达国已被川于如N...加'1物从因?..     

紫杉醇生物合成相关酶基因的克隆与表达

以牛儿基牛儿基二磷酸为前体的紫杉醇生物合成大约有20步酶促反应,其反应过程已基本阐明,近一半的相关酶基因已得到克隆与表达。综述了编码参与紫杉醇生物合成的紫杉二烯合酶、紫杉二烯5α羟化酶、紫杉烷10β羟化酶、紫杉烷13α羟化酶、紫杉二烯5α醇O乙酰基转移酶、紫杉烷2α苯甲酰转移酶、去乙酰基巴卡亭Ⅲ10βO乙酰转移酶、3氨基3苯基丙酰转移酶和3N去苯甲酰2脱氧紫杉醇苯甲酰转移酶等9个酶基因的克隆和表达方面的研究情况,并指出随着紫杉醇生物合成的分子生物学研究的不断深入,利用分子生物技术大规模生产紫杉醇将为期不远 。     

北柴胡细胞色素P450酶基因的克隆及其植物过量表达载体的构建

目的:克隆北柴胡中可能参与柴胡皂苷生物合成的细胞色素P450酶基因,构建其过量表达载体,为通过转基因验证其功能奠定基础。方法:在454高通量测序获得5'和3'端部分cDNA序列的基础上,利用LD-PCR方法获得全长cDNA,根据全长cDNA序列设计含有酶切位点的PCR引物,利用高保真酶,以RNA反转录产物为模板PCR扩增细胞色素P450酶基因的开放读框,扩增产物与pEASY-T1 Simple载体连接,转化大肠杆菌DH5α;重组质粒pT1-P450经菌液PCR和酶切方法验证后测序,采用NCBI在线Blastx、DNAman和MEGA4软件对序列进行生物信息学分析,随后将pT1-P450的酶切产物插入双元载体pCAMBIA-SUPER 1300,菌液PCR和酶切验证重组质粒p1300-P450。结果:扩增到了北柴胡细胞色素P450酶基因BcCYP87E,构建了这一基因的过量表达载体。结论:细胞色素P450酶基因的克隆和转基因载体的构建,为后续开展转基因研究,验证其生物功能奠定了基础。     

Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC)

The phbC gene encoding the third enzyme of the poly-beta-hydroxybutyrate biosynthetic pathway, poly-beta-hydroxybutyrate polymerase, in Alcaligenes eutrophus H16 has been identified by the complementation of poly-beta-hydroxybutyrate negative mutants of A. eutrophus H16. These results demonstrate that the three enzymes of the poly-beta-hydroxybutyrate biosynthetic pathway are organized phbC-phbA-phbB. Expression of all three genes in Escherichia coli results in a significant level (50% dry cell weight) of poly-beta-hydroxybutyrate production. phbC encodes a polypeptide of Mr = 63,900 which has a hydropathy profile distinct from typical membrane proteins indicating that poly-beta-hydroxybutyrate biosynthesis probably does not involve a membrane complex.