表达昆虫特异性神经毒素AaIT基因的转基因烟草的抗虫性

经改造的昆虫特异性神经毒素AaIT基因插入植物高效表达载体得到重组质粒pNGY-2。重组质粒中AaIT基因5＇端与烟草花叶病毒(TMV)的Ω序列3＇端融合,受两个串联的35S启动子控制,通过土壤农杆菌LBA4404介导转化烟草NC89,经NPTⅡ选择后再经GUS染色挑选出阳性再生植株,Southern blotting进一步证实了AaIT基因已经整合到烟草基因组中,对棉铃虫(Heliothis armigera)的抗虫实验表明,转基因烟草有显著的抗虫活性。     

抗性库蚊酯酶基因在大肠杆菌中的克隆和表达

用抗性库蚊酯酶基因,引入原核表达载体pRL439,转化大肠杆菌HB101细胞,获得表达。通过酶切、Southern杂交鉴定重组质粒。研究了重组菌酯酶的活性,重组质粒pRLB1表达的酯酶具有高酶活并能高效降解酯酶的特异性底物α乙酸萘酯(αNA)和β乙酸萘酯(βNA);经对重组菌进行细胞固定化后降解农药三氯杀虫酯(7504),反应时间短,降解效率高     

蝴蝶兰LFY基因克隆及高效植物表达载体的构建

根据NCBI中蝴蝶兰LFY花序分生组织基因序列设计2对引物,用RT-PCR法从蝴蝶兰花芽中扩增出LFY基因,对扩增产物进行克隆和测序.结果表明,获得的蝴蝶兰LFY基因约为1500 bp,与报道序列同源性达98.71％.将LFY基因插入pRI101-ON载体中,经PCR、双酶切及测序鉴定,证实重组表达质粒中含有目的片段,表明成功构建了高效植物表达载体pRI1O1-LFY.     

小鼠白细胞介素4基因的化学合成、克隆与表达

利用381A型DNA合成仪,分29个寡聚核苷酸片段化学合成了小鼠IL-4全基因,共442bp。以pUC12质粒作为载体,将所有合成片段分前后两组进行磷酸化、退火、连接和克隆,经过菌落原位杂交、酶切鉴定和质粒DNA序列分析,分别得到了含有小鼠IL-4前后两半基因片段的两种重组质粒,回收前半基因片段,插入到含有后半基因重组质粒的EcoRI和PstI酶切位点之间,成功地得到了含有小鼠IL-4全基因的重组质粒pFR101。将全合成基因插入到质粒pSM53中,得表达质粒pFR105,转化大肠杆菌TAP106,根据IL-4对CTLL细胞的作用,肯定了TAP106(pFR105)细菌中有小鼠IL-4活性蛋白的表达。     

绿色荧光蛋白基因(GFP)在抗虫转基因植物研究中的应用(英文)

用合成的cry1Ac基因与绿色荧光蛋白基因 (GFP)构成融合蛋白基因 ,然后和改造的GNA基因构建双价抗虫基因植物表达载体pBGbfg ,经根癌农杆菌介导转化了烟草。在紫外灯照射下 ,观察到转基因植株叶片中有较强的绿色荧光 ;经抗虫试验、PCR、Southernblot和Westernblot等检测 ,表明该重组植物表达载体能够在转基因植物中有效表达外源基因 ,转基因植株绿色荧光的表型与其抗虫性密切相关。从而成功地建立了以绿色荧光蛋白基因与抗虫基因组成的融合基因转化系统 ,简化了抗虫转基因植物筛选程序 ,有助于快速获得双价抗虫转基因植株。     

绿色荧光蛋白基因（G卯）在抗虫转基因植物研究中的应用

用合成的crylAc基因与绿色荧光蛋白基因(GFP)构成融合蛋白基因,然后和改造的GNA基因构建双价抗虫基因植物表达载体pBGbfg,经根癌农杆菌介导转化了烟草。在紫外灯照射下,观察到转基因植株叶片中有较强的绿色荧光;经抗虫试验、PCR、Southern blot和Western blot等检测,表明该重组植物表达载体能够在转基因植物中有效表达外源基因,转基因植株绿色荧光的表型与其抗虫性密切相关。从而成功地建立了以绿色荧光蛋白基因与抗虫基因组成的融合基因转化系统,简化了抗虫转基因植物筛选程序,有助于快速获得双价抗虫转基因植株。     

高光效基因植物表达载体的构建

通过一系列中间载体的构建 ,将来自C4作物 (甘蔗 )光合作用中的关键酶基因 (高光效基因 )RbcS和PEPCase基因插入到植物表达质粒中 ,获得了具有卡那霉素抗性的RbcS植物表达载体 pC2 0SNR和具有PPT除草剂抗性的PEPCase基因的正义 (pC30SNP)和反义 (pC30SNPr)植物表达载体。再将这 3个载体分别转入农杆菌LBA4 4 0 4和A2 81或EHA10 5中 ,获得了适于C3 植物转化的农杆菌重组工程菌株。为进一步利用C4植物高光效基因转化C3 植物 ,以期提高光合效率提供基础。     

含人抗体恒定区基因的酵母表达质粒的构建

本文主要报告了用以表达人-鼠嵌合抗体基因的酵母表达载体的构建、筛选与鉴定的研究结果.作者利用高度表达的酵母质粒YEP_(51),与含人抗体恒定区基因的pSV_2gptHuG_4两个质粒为材料,构建成可以供表达任何小鼠单克隆抗体的可变区基因,以构成嵌合的人-鼠抗体基因,用酶切回收插入DNA片段、原位和斑点杂交法对重组质粒予以鉴定和筛选,获得了pYEP-H重组的酵母表达载体.     

几丁质酶表达质粒pKChiA和pMChiA的构建及表达

从质粒pLCHIA中切出含粘质沙雷氏菌（Serratia marcescens）几丁质酶基因（chiA）的1.8kb HinfI片段,将其插入到表达载体pKK223-3强启动子Ptac下游的SmaI位点内,构建成几丁质酶表达质粒pKChiA。再用内切酶BamHI从pKChiA质粒中切出2.1kb Ptac-ChiA片段,并将其重组到质粒pMC71A的单一BamHI位点内,构建成另一种几丁质酶表达质粒pMChiA。这两种质粒可在大肠杆菌HB101和JM105中高效表达,几丁质酶表达水平比质粒pLCHIA高1－3倍。     

苏云金杆菌6－内毒素基因及3’末端缺失基因在大肠杆菌和农杆菌中的亚克隆和表达

苏云金芽孢杆菌交种Bacillus thuringiensis aizawai 7-29 δ-内毒素基因及其3,末端缺失基因,亚克隆到质粒pUCl9上,构建了Pucf33和Puck63重组质粒。进一步将δ-内毒素基因及其3’端缺失基因,插入到植物表达载pBl121．2质粒中,构建了Pgy61和Pgyck63重组质粒。．用32P标记的δ-内毒素3’末端缺失的KpnI DNA片段的探针,与重组质粒进行DNA—DNA分子杂交,结果阳性。带有全长和3’末端缺失重组质粒,转入大肠杆菌HB101和农杆菌LA4404受体细胞中,其克隆子细胞抽提物,对大菜粉蝶和玉米螟幼虫均具有杀虫生物活性。结果证明δ－内毒素基因不仅能在大肠杆菌HB10l中,而且能在农杆菌LA4404中表达。     

苎麻CCoAOMT基因cDNA反义转化模式烟草''WS38''

苎麻咖啡酰辅酶A氧甲基转移酶(CCoAOMT)是其木质素合成过程的一种关键酶,运用克隆的该酶基因cDNA及植物表达载体pBI121、pWM101,分别构建了35S启动子控制的苎麻CCoAOMT基因反义cDNA基因质粒(pBI121-antiBnCCoAOMT)和cDNA全长表达质粒(pWM101-BnCCoAOMT),并通过根癌农杆菌介导法将其转化至模式烟草WS38,获得了转基因烟草.对转基因植株进行分子分析和组织学初步研究表明,转反义RNA基因植株叶柄木质素含量较野生烟草或转正义基因烟草叶柄木质素含量降低.说明运用反义RNA技术对CCoAOMT基因的表达进行基因工程调控,一定程度上可以对木质素的合成产生干扰,为获得低木质素或木质素组分改良的苎麻基因工程奠定基础.     

Isolation, characterization, and sequence analysis of cryptic plasmids from Acinetobacter calcoaceticus and their use in the construction of Escherichia coli shuttle plasmids.

Three cryptic plasmids have been discovered in Acinetobacter calcoaceticus BD413. These three plasmids, designated pWM10 (7.4 kb), pWM11 (2.4 kb), and pWM12 (2.2 kb), exhibited extensive homology to one another, as shown by Southern blot hybridization and restriction site analysis data, and also hybridized with three plasmids having slightly different sizes detected in a second strain, A. calcoaceticus BD4. Plasmid pWM11 and a fragment of pWM10 were each subcloned into pUC19, yielding plasmids pWM4 and pWM6, respectively, and were used in a series of inter- and intraspecies transformation experiments. Both plasmids replicated as high-copy-number plasmids in A. calcoaceticus BD413, as well as in strains of Escherichia coli. However, when transformed into the oil-degrading strain Acinetobacter lwoffii RAG-1, both plasmids were maintained at low copy numbers. No modification of the plasmids was detected after repeated transfers between hosts. An analysis of a series of deletions demonstrated that (i) a 185-bp fragment of pWM11 was sufficient to permit replication of the shuttle plasmid in A. calcoaceticus BD413, (ii) the efficiency of transformation of A. calcoaceticus BD413 decreased according to the size of the deletion in the insert by up to 4 orders of magnitude, and (iii) the entire insert was required for transformation and replication in A. lwoffii RAG-1. The sequence of pWM11 contained several small (150- to 300-bp) open reading frames, none of which exhibited any homology to known DNA or protein sequences. In addition, a number of inverted and direct repeats, as well as six copies of the consensus sequence AAAAAAATA previously described for a cryptic plasmid from A. lwoffii (M. Hunger, R. Schmucker, V. Kishan, and W. Hillen, Gene 87:45-51, 1990), were detected. Cloning and expression of the alcohol dehydrogenase regulon from A. lwoffii RAG-1 were accomplished by using the Acinetobacter shuttle plasmid.     

抗虫双价基因表达载体的构建及微束激光转化植物研究

将苏云金芽孢杆菌伴孢晶体(Bacillus thuringiensis,Bt)蛋白的基因与反枝苋菜凝集素(Amaranthus retrojflexus agglutinin,ARA)基因构建成双价基因的表达载体,编码一种既能抗鳞翅目害虫,又能抗同翅目蚜虫的杀虫蛋白.把双价基因(Bt-BA)连接到原核表达载体pET28a和植物表达载体pBI121上,经过限制性酶切分析和PCR鉴定,结果表明:含有双价基因的原核和真核重组表达质粒均已构建成功.将该双价基因转入油菜,获得抗性小植株.     

嗜碱细菌NTT36嗜碱基因的亚克隆及其在大肠杆菌和农杆菌中的表达

将大肠杆菌ＨＢ１０１嗜碱转化子中质粒ｐＧＣＡ所携带的嗜碱基因亚克隆至双元载体ｐＢＩ１２１质粒中,构建了植物表达载体ｐＬＧＣ重组质粒。用其转化大肠杆菌ＨＢ１０１获得了能在碱性和卡那霉素抗性平板上生长的转化子,再通过三亲交配法将亚克隆质粒ｐＬＧＣ转化进农杆菌ＬＢＡ４４０４,又获得能在碱性平板和卡那霉素及利福平双抗平板上生长的转化子,Ｓｏｕｔｈｅｒｎ杂交结果表明ＨＢ１０１转化子亚克隆质粒ｐＬＧＣ是由来自于嗜碱芽孢杆菌ＮＴＴ３６染色体ＤＮＡ和双元载体ｐＢＩ１２１组成,且农杆菌ＬＢＡ４４０４转化子含有来自大肠杆菌亚克隆转化子的ｐＬＧＣ质粒。     

Characterization of Bacteroides ovatus plasmid pBI136 and structure of its clindamycin resistance region.

Genetic and physical analyses were used to characterize the Bacteroides ovatus R plasmid pBI136. Results from restriction endonuclease cleavage studies were used to construct a physical map of the plasmid for the enzymes EcoRI, BamHI, ClaI, XbaI, SalI, and SmaI. Based on the sizes of restriction fragments generated in these studies, the plasmid was estimated to be 80.6 kilobase pairs (kb). A 7.2-kb region of the plasmid required for resistance to lincosamide and macrolide (LM) antibiotics was mapped by analysis of spontaneously occurring LM-sensitive deletion derivatives. Hybridization studies showed that this region and an adjoining 2.9-kb EcoRI fragment were responsible for the previously reported homology among Bacteroides plasmids pBF4, pBFTM10, and pBI136. Within this region of homology, 0.5 kb was attributed to a directly repeated sequence thought to bound the LM resistance determinant on pBF4 and pBFTM10. Two pBI136 EcoRI fragments spanning the putative LM resistance region were cloned in Escherichia coli, and heteroduplex analysis of these recombinant plasmids revealed the presence of a 1.2-kb directly repeated sequence. These results suggested that the pBI136 LM resistance determinant resides on an 8.4-kb segment of DNA containing 6.0 kb of intervening DNA sequences bounded by a 1.2-kb directly repeated sequence.     

Large transmissible clindamycin resistance plasmid in Bacteroides ovatus.

Bacteroides ovatus IB106 contained two plasmids, pBI106 (46 kilobases) and pBI136 (82 kilobases). Transmissible clindamycin-erythromycin resistance (Ccr) was mediated by pBI136 , whose Ccr determinant was closely related to the determinant on the Bacteroides R plasmids pBF4 and pBFTM10 . Hybridization studies showed that pBI106 was not involved in Ccr transfer, but it shared extensive homology to pBF4 with the exception of the pBF4 region implicated in Ccr.     

Development and use of cloning systems for Bacteroides fragilis: cloning of a plasmid-encoded clindamycin resistance determinant.

Chimeric plasmids able to replicate in Bacteroides fragilis or in B. fragilis and Escherichia coli were constructed and used as molecular cloning vectors. The 2.7-kilobase pair (kb) cryptic Bacteroides plasmid pBI143 and the E. coli cloning vector pUC19 were the two replicons used for these constructions. Selection of the plasmid vectors in B. fragilis was made possible by ligation to a restriction fragment bearing the clindamycin resistance (Ccr) determinant from a Bacteroides R plasmid, pBF4;Ccr was not expressed in E. coli. The chimeric plasmids ranged from 5.3 to 7.3 kb in size and contained at least 10 unique restriction enzyme recognition sites suitable for cloning. Transformation of B. fragilis with the chimeric plasmids was dependent upon the source of the DNA; generally 10(5) transformants micrograms-1 of DNA were recovered when plasmid purified from B. fragilis was used. When the source of DNA was E. coli, there was a 1,000-fold decrease in the number of transformants obtained. Two of the shuttle plasmids not containing the pBF4 Ccr determinant were used in an analysis of the transposon-like structure encoding Ccr in the R plasmid pBI136. This gene encoding Ccr was located on a 0.85-kb EcoRI-HaeII fragment and cloned nonselectively in E. coli. Recombinants containing the gene inserted in both orientations at the unique ClaI site within the pBI143 portion of the shuttle plasmids could transform B. fragilis to clindamycin resistance. These results together with previous structural data show that the gene encoding Ccr lies directly adjacent to one of the repeated sequences of the pBI136 transposon-like structure.     

Molecular cloning of a gene coding for a Vibrio cholerae haemagglutinin

Recombinant plasmids encoding a Vibrio cholerae haemagglutinin were isolated from the highly virulent V. cholerae strain C5 by cosmid cloning. Both Escherichia coli HB101 containing the recombinant plasmids and V. cholerae C5 were able to agglutinate a variety of erythrocytes from human and animal origin; this haemagglutination was not inhibited by D-mannose or L-fucose. Subcloning of the recombinant cosmid DNA revealed that a 1.3 kb DNA fragment was sufficient for haemagglutinin production in E. coli HB101. Under direction of this 1.3 kb Vibrio DNA fragment, two proteins were made in E. coli minicells, of 27 and 10 kDa. Haemagglutinin-encoding sequences were not detected in every V. cholerae strain.     

Characterization of DNA fragments encoding fimbriae of the uropathogenic Escherichia coli strain KS71

Recombinant plasmids were constructed that expressed the KS71A, KS71B and KS71C fimbrial antigens of the pyelonephritogenic Escherichia coli strain KS71 (O4:K12) in E. coli HB101. The KS71C-encoding genes were located on a 6.4 kb HindIII-XhoI fragment obtained from the recombinant cosmid pKTH145 that expresses this antigen. Spontaneous KS71C-mutants were isolated that contained a 0.8 kb insert in a specific restriction fragment of KS71C-encoding recombinant plasmids. The KS71B-encoding segment was located on a 11.5 kb deletable DNA fragment of recombinant cosmid pKTH144. A DNA fragment encoding the KS71A fimbria was obtained on a 12 kb EcoRI fragment of the recombinant cosmid expressing this antigen in E. coli HB101 and closely resembled the KS71B-encoding fragment. In the recombinant cosmid, the KS71B-expressing region was flanked by homologous DNA segments. A similar stretch of DNA was found close to the KS71A-expressing DNA region.     

Modified binary plant transformation vectors with the wild-type gene encoding NPTII.

The defective gene encoding neomycin phosphotransferase (NPTII) present in the binary plasmid vector, pBin19, was replaced with the wild-type (wt) gene. Plasmid vectors analogous to pBin19, pBI121 and pBI101 were constructed carrying the gene encoding the wt NPTII enzyme activity.