转乙肝病毒表面抗原基因烟草发根的获得

构建了乙肝病毒表面抗原基因（HBsAg）植物表达载体,通过冻融法将HBsAg 基因转入到发根农杆菌LBA1314中,采用叶盘法将HBsAg基因导入到烟草中,获得了转基因烟草发根.对转基因烟草发根的GUS检测结果表明：转HBsAg基因烟草发根可以染成蓝色,而非转基因烟草的根没有染色反应.这说明gus基因在转HBsAg基因烟草发根获得了表达.     

根癌农杆菌法转化烟草的条件探索

 利用含有四环素阻遏蛋白,具有卡娜霉素抗性的烟草种子为实验材料,采用根癌农杆菌介导转化中的叶圆盘法,将外源基因导入烟草愈伤组织,建成可诱导表达细胞质和细胞核CaM/CaM结合多肽的转基因烟草体系.在获得转基因植物的过程中,通过比较影响根癌农杆菌转化频率的各种因素,表明植物材料的选择和培养,农杆菌的培养和稀释,植物材料与农杆菌共培养的程度及转基因植株的筛选条件是影响农杆菌转化效率的重要因素.     

分泌性表达人rPA毕赤酵母工程菌株的构建及发酵条件的初步优化

通过PCR扩增得到组织型纤溶酶原激活剂突变体Reteplase(rPA)基因片断，将该基因片断插入到含有AOX1启动子和α分泌信号肽序列载体pPIC9K中，构建了重组表达质粒pPICgK／rPA，转化Pichia pastoris GS115宿主菌．通过比较转化子在MM和MD平板上的生长状况，筛选His^ Mut^s表型转化子．并在2．0mg/mL G418平板筛选得到多拷贝转化子GR10，GR11．摇瓶培养，甲醇诱导外源基因表达，表达产物经SDS-PAGE和Western blot分析，结果表明重组蛋白分子量约39ku，能与特异性单克隆抗体发生免疫反应；体外气泡法测定rPA活性，结果显示重组蛋白具有较好的纤溶活性．通过正交试验，优化发酵条件，表达活性最高为1050IU／mL．     

TBSV病毒瞬时表达载体构建及其表达

为了深入理解植物RNA病毒载体表达系统,初步建立了利用TBSV病毒表达载体在寄主植物烟草中表达外源蛋白的技术体系.以番茄丛矮病毒(Tomato bushy stunt virus,TBSV)为材料,构建了含报告基因sGFP的WY1,WY2等2个重组病毒表达载体,并将载体导入农杆菌GV3101用于侵染烟草植株叶片;利用农...     

抗人端粒酶逆转录酶单链抗体基因在烟草中的表达

构建了含抗人端粒酶逆转录酶单链抗体基因ScFv-hTERT的植物表达载体p1304-SH,通过农杆菌介导的叶盘法转化烟草.转基因烟草植株叶片总DNA的PCR,Southern b lot检测结果表明,ScFv-hTERT基因已整合进了转基因烟草植株基因组中;RT-PCR,SDS-PAGE分析证实目的基因已在烟草叶片中成功表达,竞争性ELISA的结果表明,表达的重组抗体与其抗原有良好的结合活性.     

农杆菌介导的大豆反义fad2-1基因转化烟草研究

以烟草无菌苗叶片为外植体,通过根癌农杆菌介导法,将大豆反义油酸脱饱和酶基因导入烟草中,经梯度卡那霉素筛选,获得可在含75mg/L卡那霉素选择生根培养基上再生的抗性植株75株,其中67株抗卡那霉素植株的PCR分析扩增出目的片段;RT-PCR分析表明该基因在烟草中表达.结果表明大豆反义油酸脱饱和酶基因在烟草基因组中整合和表达.这为大豆反义油酸脱饱和酶基因转入大豆和表达奠定基础,也为烟草基因工程育种提供了依据.     

异戊烯基转移酶基因的克隆与转化烟草的研究

从根癌农杆菌中克隆了异戊烯基转移酶基因,构建了植物表达载体p35S-ipt(pVCT2131).用此载体转化烟草,结果表明,ipt在CaMV 35S启动子的控制下,可以大大提高烟草愈伤组织诱导率.     

苦瓜McAG2启动子的克隆与表达研究

文中从苦瓜基因组中克隆得到长为1417 bp的McAG2基因5′上游片段并进行了DNA序列分析.通过PCR得到了其缺失片段,将其插入pBI121载体替换CaMV35S启动子,得到了McAG2基因5′侧翼缺失表达载体.并利用农杆菌介导转化烟草,建立了相应的转基因烟草植株,以研究其在不同器官组织中的表达特性. β-glucuronidase(GUS)染色结果显示该启动子在转基因烟草叶片和根组织中没有表达活性.     

复苏植物牛耳草BhLEA2基因启动子的分离和基因表达模式研究

为研究干旱诱导的基因表达模式,利用Inverse-PCR和Tail-PCR的方法从复苏植物牛耳草（Boea hygrometrica（Bunge）R Br．）中扩增出胚胎发育晚期丰富蛋白编码基因BhLEA2的1215bp启动子序列,命名为PBhLEA2。构建了由PBhLEA2启动子引导GUS嵌合基因的植物表达载体pBhLEA2-GUS,并经农杆菌介导导入到烟草中,得到4株转基因烟草植株。GUS检测分析表明,该启动子可驱动GUS报告基因在转基因烟草的种子和刚萌发的小苗的子叶和胚轴中高效表达,在10-20d苗龄的植株中不表达,表现出一定的发育阶段和组织特异性。干旱、高盐胁迫及脱落酸（ABA）、乙烯和H2O2等信号分子可在不同程度上诱导GUS报告基因在20d苗龄的转基因植株的叶片中表达,但只有ABA可诱导其在根中表达,表明该启动子对BhLEA2基因表达的调控受环境信号影响。     

小鼠液胞H~+-ATPase 15 K启动子(M15 K)的克隆及其在植物体内的表达特性研究

PCR克隆了小鼠液胞H+-ATPase 15K启动子,构建具有Kan抗性和GUS intron报告基因的植物表达载体LpPMG.通过M15K启动子指导的GUS intron基因在烟草叶片内的瞬时性表达,比较了其植物表达特性.结果表明:M15K启动子可启动GUS在植物体内的表达.其表达活性相当于2×35S启动子的87.0%±17.3%.     

Obtaining High Pest-resistant Tobacco Plants Carrying B.t. insecticidal Gene

To increase the expression level of CryIA(c) gene in transgenic plants, a plant expression vector pBinMoBc carrying the CryIA(c) gene under control of chimeric OM promoter and Ω factor was constructed. As a control, pBinoBc carrying the CryIA(c) gene with the CaMV 35S promoter was also constructed. The vectors were transferred into tobacco plants respectively via Agrobacterium-mediated transformation. ELISA assay showed that the expression level of the CryIA(c) gene in pBinMoBc transgenic tobacco plants was 2.44-times that in pBinoBc transgenic tobacco plants, and it could be up to 0.255% of total soluble proteins. Bioassay showed that pBinMoBc transgenic tobacco plants had more notable insecticidal effect than pBinoBc transgenic tobacco plants. The above results showed that the chimeric OM promoter was a stronger promoter than CaMV 35S promoter that was widely used in plant genetic engineering, and this is very useful in pest-resistant plant genetic engineering.     

绿色荧光蛋白基因在烟草植株中的表达

用冻融法将含有绿色荧光蛋白基因的质粒ｐＧＬ２－ＧＳ导入农杆菌菌株ＬＢＡ４４０４ （ｐＴＯＫ２３３），两个质粒发生同源重组，形成一个新的二元载体ｐＴＯＫ２３３－ＧＳ．农杆菌菌株ＬＢＡ４４０４ （ｐＴＯＫ２３３－ＧＳ） 经叶盘法转化烟草，筛选具有卡那霉素抗性的愈伤组织，诱导成苗．ＰＣＲ 扩增发现，绿色荧光蛋白基因在９０％ 的再生植株中存在．在荧光显微镜下，使用蓝色激发光观察再生植株的徒手切片和压片发现，８０ ％ 以上的再生植株都能不同程度地发出绿色荧光．多数植株的根尖和叶脉都发光，一些植株的气孔、叶表皮或叶绒毛也发光     

Transgenic tobacco plants harboring tomato proteinase inhibitor II gene and their insect resistance

The plant expression vectors pBCT2 and pBT2 were constructed with the cDNA sequence (tin2) and genomic DNA sequence (tin2i) of tomato proteinase inhibitor II gene respectively. Then the two expression vectors were transferred into tobacco via the Agrobacterium tumefaciens strain LBA4404, and transgenic tobacco plants were generated. Molecular analysis and trypsin activity assay showed that both cDNA and genomic DNA were expressed properly in the transgenic plants. Insecticidal activities in these transgenic plants indicated that transgenic tobacco plants carrying tin2i sequence were more resistant to 2-instar larvae of Heliothis armigera Hubner than those carrying tin2 sequence. Therefore the intron of tin2i sequence might be a contributor to insecticidal activity of the transgenic tobacco.     

苜蓿花叶病毒复制酶基因全长cDNA植物表达载体的构建和对烟草的转化

将苜蓿花叶病毒中国分离株(Alfalfa mosaic virus Chinese isolate，A1MV-Ch)的复制酶P2亚基(90 kD蛋白)基因的全长cDNA构建到植物表达载体pROKⅡ中，得到重组植物表达载体pAIMV-FL．用三亲融合法导入农杆菌LBA4404，并转化烟草，经PCR检测，获得了含全长cDNA的转基因烟草植抹．     

Expression of two insect-resistant genes crylA ( b&c )/GNA in transgenic tobacco plants results in added protection against both cotton bollworm and aphids

The synthesized Bacillus thuringiensis insecticidal protein gene crylA(b&c) and the synthesized gene GNA, (the mannose specific lectin from snowdrop ( Galanthus nivalis)), tumefaciens have been inserted into plant expression vector pGW4BAI. Leave stripes of Nico-tiana tabacum var. K326 have been transformed with Agrobacterium tumefaciens strain LBA4404 harboring the plant expression vector. 28 kanamycin resistant tobacco plants have been obtained. PCR and Southern blot analyses show that the foreign crylA and GNA genes have been inserted into the genome of transformed tobacco plants. Haemagglutination assays show that GNA has a functional activity. Leaf disc bioassays against cotton bollworm ( H. armigera) show that the transgenic tobacco plants have a high insecticidal activity. The inhibition of aphid population in leaf disc bioassays against Myzus persicae shows that the fecundity of aphid on transgenic plants is lower than that on untransformed plants; the aphid population on the transgenic tobacco plants is 25%-70% that on untransformed tobacco plants. ELISA analysis of CrylA protein in tobcco leaves provides similar data to bioassay results. Through the two bioassays against H. armigera and M. persicae, several transgenic tobacco plants showing high insect-resistant activities to both pests have been obtained.     

利用人内皮抑素基因构建植物表达载体及对烟草的遗传转化

将人内皮抑素(endostatin)基因重组于植物双元表达载体pGA 643,得到重组质粒pGE.用三亲融合法将其导入农杆菌LBA 4404中,采用叶盘法转化烟草,经诱导与筛选,获得了卡那霉素抗性植株.提取抗性植株总DNA,通过PCR扩增和Sou thern杂交检测,筛选出了整合有外源基因的转化植株.为进一步研究人内皮抑素在烟草中的表达及生物学功能奠定了基础.     

胰蛋白酶抑制剂抗虫基因转化烟草的研究

实验用携带质粒pAM194/TI的根癌农杆菌LBA4404转化烟草,通过筛选,共获得35株卡那霉素抗性苗.GUS组织染色、PCR检测及抗虫试验表明胰蛋白酶抑制剂抗虫基因已经成功地整合进再生植株染色体基因组中,有些已正确表达.     

根癌农杆菌介导苜蓿体胚转化及转基因植株再生

用含质粒载体pCAMBIA2301(带有受CaMV35S启动子调控的GUS基因和nptⅡ基因)的根癌农杆菌Agrobacterium tumefaciens转化晋南苜蓿Medicago sativa L.cv.Jinnan的体胚组织,发现负压处理有利于提高转化频率(可达35%),3批共158个体胚切块的转化实验共获得具有卡那霉素抗性的再生植株15株,经组织化学染色和分子检测,证实GUS基因已整合到转化植株基因组中,在芽、叶片、叶柄和根等组织中均有表达,并在土壤栽培过程中保持稳定的表达.