TaNHX2基因植物表达载体的构建及在烟草中的表达

将TaNHX2基因重组于质粒pBIN438的CaMV 35S启动子下游,构建含TaNHX2基因的植物双元表达载体pBIN438-TaNHX2.采用农杆菌介导转化烟草(Nicotiana tobacum L.),获得含TaNHX2的转基因烟草植株.经PCR、RT-PCR分析表明,TaNHX2基因已整合到烟草中,并且得到表达.耐盐分析表明,外源基因TaNHX2提高了转基因植株的耐盐性.     

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.     

Function of resveratrol derived from transgenic plant expressing resveratrol synthase gene

Two genes from grapevine coding for resveratrol synthase, named RS1 and RS2, were cloned by RT-PCR. AnEscherichia coli expression vector was constructed by insertion of RS1 into pBV221. A specific protein with the same molecular weight (42 ku) as the resveratrol synthase was expressed and used to prepare the rabbit antiserum. A plant expression vector was constructed by inserting the RS1 gene into pBin438 downstream of the doubled CaMV 35S promoter and TMV-Ω fragment. PCR-positive transgenic tobacco plants were obtained after transformation withAgrobacterium tumefaciens LBA4404 harboring the plant expression vector. Southern blot analysis demonstrated that the foreign gene was integrated into the tobacco genome. The results of RT-PCR and Western blot indicated that the RS1 gene was transcribed and expressed. Formation of resveratrol in transgenic tobacco was further determined by thin-layer chromatography of silica gel and HPLC. Increased accumulation of human breast adenocarcinoma cells in G₀ and G₁ phases of cell cycle was observed in cells treated with resveratrol purified from transgenic tobacco as compared to the untreated cells.     

表达长喙田菁SrPCS4烟草的获得及Cd抗性研究

植物螯合肽(phytochelatins,PCs)在植物解除重金属的毒性方面具有重要作用,是以谷胱甘肽为底物,在植物螯肽合成酶(phytochelatin synthase,PCS)催化下合成的.作者已经克隆得到的长喙田菁(Sesba-nia rostrata)植物螯合肽合成酶SrPCS4 cDNA长为1035 bp,其ORF编码177个氨基酸,以pHANNIBAL及pART27为基础,构建了CaMV35S启动子驱动的SrPCS4基因植物表达载体pAM25,采用电击转化方法将pAM25导入根癌农杆菌EHA105,并通过改良叶盘转化方法用该菌株对烟草进行了转化,对转基因烟草进行了PCR与northern-blot检测,研究结果表明得到了表达该基因的烟草,但表达该基因的烟草不能够提高对Cd的抗性.     

利用番茄U3snRNA基因上游启动区构建植物表达载体及对烟草的转化

利用番茄U3snRNA基因上游启动区和ACC合成酶反义RNA-核酶嵌合基因DNA片段,构建含U3snRNA基因上游启动区-ACC合成酶的反义RNA-核酶嵌合序列的表达载体,重组于植物双元表达载体pGA643中,得到pGU3R.用三亲融合法导入农杆菌LBA4404中,采用叶盘法转化烟草,诱导再生小植株,获得了卡那霉素的抗性植株.提取抗性植株总DNA,通过PCR、PCRSouthern杂交检测并分别用启动区序列和ACC合成酶的反义RNA-核酶嵌合序列作探针,通过Southern杂交检测,已筛选出整合有外源基因的转化植株.为进一步研究U3snRNA上游启动区增强反义RNA-核酶基因的表达奠定了基础.     

毛白杨4CL基因启动子的克隆及初步功能分析

4CL(4-coumarate:CoA ligase,4-香豆酸:辅酶A连接酶)在植物木质素合成途径中催化羟基香豆酸生成羟基肉桂酰CoA,主要在木质部中表达,对植物木质素生物合成具重要调控作用.为研究4CL基因启动子在转基因植物中的表达特性,探索其在植物基因工程研究中的潜在应用价值,利用PCR方法从毛白杨基因组DNA中扩增得到了4CL启动子片段.序列分析表明与美洲山杨(P.tremuloids)的4CL启动子同源性为95%.采用生物信息学方法对该序列进行分析.与GUS基因融合构建双元表达载体,转化烟草的瞬时表达检测可见明显GUS活性.     

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.     

Regulatory effect of cotton leaf curl virus AC2 on virion sense promoter

The AC2 gene of cotton leaf curl virus (CLCuV) was obtained by polymerase chain reaction (PCR) . The total DNA of the CLCuV infected tomato leaves was used as template, and the amplified DNA fragment was inserted into a cloning vector. Transient expression vectors were constructed by inserting the AC2 gene into downstream region of CaMV 35S promoter. These constructs were delivered into tobacco and cotton leaf cells for transient expression by particle bombardment. The results indicated that the virion sense promoter was activated by AC2 and its activity increased remarkably. However, the activity of transactivated virion sense promoter was still lower than that of the complementary sense promoter. The expression pattern of transactivated virion sense promoter was similar to that of the complementary sense promoter, namely with high activity in both mesophyll and vascular tissues. The possibility of application of AC2 in plant genetic manipulation was also explored.     

Enhanced late blight resistance of transgenic potato expressing glucose oxidase under the control of pathogen-inducible promoter

To engineer crop disease resistance by utilizing natural defense mechanism that was expressed in the incompatible host-pathogen interactions is expected to result in a durable and broad-spectrum resistance. In order to prove this viewpoint, we amplified the coding region of the glucose oxidase (GO) gene from Aspergillus niger via PCR and fused it to the pathogen-inducible promoter, Prp1-1. The chimeric gene was cloned into a plant expression vector and conjugated into Agrobacterium. Twenty-three transgenic potato plants were obtained by Agrobacterium-mediated transformation. The integration of GO gene was confirmed by Southern hybridization and the GO gene expression was identified with KI-starch color reaction. Phytophthora infestans inoculation revealed that the expression of the chimeric transgene was induced by pathogen infection. Most of the transgenic plants exhibited various degrees of enhanced disease resistance. Four of them had lesion sizes reduced to less than half of the non-transgenic controls. One plant showed disease resistance of the hypersensitive response. These results testified the feasibility of our strategy of expressing GO transgene under the control of the disease-inducible promoter in engineering crop disease resistance.     

Establishment of a coupled expression system mediated by modified T7 RNA polymerase gene

A coupled expression system for plants wasmerase gene was modified by addition of the coding sequenceof nuclear location signal from SV40 large T antigen. Plantexpression vector pBBT7 was constructed with the modifiedT7 RNA polymerase gene under the control of CaMV35Spromoter. Another expression vector pBTG contained cas-sette of gusA controlled by T7 promoter. The two vectorswere co-transformed into tobacco via the Agrobecte-rium-mediated method. Results of GUS activity indicatedthat the co-transformed plant with pBBT7 and pBTGshowed a high level of GUS activity. The results demon-strated that the coupled expression system of T7 polymeraseand T7 promoter was workable in plants.     

Analysis of the Cotton E6 Promoter

An E6 gene from sea island cotton （Gossypium barbadense） was expressed specifically in cotton fiber cells to transfer functions to cultivated species for better transgenic engineering. The regulatory activity of the E6 promoter region was then studied by isolating a 614-bp fragment of the 5＇-flanking region from upland cotton （Gossypium hirsutum CR1-12） to produce a green fluorescent protein （GFP） reporter construct for analysis of tissue-specific expression in transgenic tobacco seedlings. Fluorescent analyses indicate that the relatively short E6 promoter is sufficient to direct green fluorescent protein expression specifically in the leaf trichomes （hair cells） of the transgenic tobacco plants. As cotton fibers are also unicellular trichomes that differentiate from epidermal cells of developing cotton ovules, the result suggests that the relatively short E6 promoter can serve as a fiber-specific expression promoter for genetic engineering to improve cotton fiber quality.     

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%.     

Construction of a new plant expression vector containing two insect resistant genes and its expression in transgenic tobacco plants

A new plant expression vector (pBS29K-BA) containing two insect resistant genes, a synthetic chimeric gene BtS29K encoding the activated insecticidal protein Cry1Ac and a gene API-BA encoding the arrowhead (Sagittaria sagittifolia L.) proteinase inhibitor (API) A and B, is constructed. Transgenic tobacco plants expressing these two genes are obtained through Agrobacterium-mediated transformation of tobacco leaf discs. The average expression levels of Cry1Ac and API-BA proteins in transgenic plants are of 3.2 μg and 4.9 μg per gram fresh leaf respectively. The results of insecticidal assay of transgenic plants indicate that the pBS29K-BA transformed plants are more resistant to insect damage than the plants expressing the Cry1Ac gene or API-BA gene alone.     

Obtaining transgenic rice resistant to rice fungal blast disease by controlled cell death strategy

The strategy of the two-component system,composed of Barnase and Barstar which encode RNase and a specific inhibitor to the RNase respectively, is adopted to obtain transgenic rice resistant to rice fungal blast disease. In this study, two chimeric promoters, induced by rice blast fungus pathogen (Magnaporthe grisea), are fused with Barnase respectively to construct two plant expression vectors, pWBNBS and pPBNBS together with the Barstar driven by CaMV 35S promoter. The resistance of the transgenic rice lines to rice blast fungus disease and rice blight disease are evaluated. The results show that (1) the expression of Barnase is induced in rice leaves when inoculated with the spores of Magnaporthe grisea; (2) the induced expression level of Barnase surpasses the level of Barstar, which elicits a similar hypersensitive response (HR) in the leaves, and the transgenic plant shows high resistance to the rice fungal blast disease; and (3) transgenic rice plants also show obvious resistance to rice bacterial blight disease. Taken together, these results suggest that the transgenic rice plants harboring this two-component system acquire relatively broad spectrum resistance against pathogens, especially high resistance to rice fungal pathogen.     

转蔗糖: 蔗糖-1-果糖基转移酶基因提高烟草的耐旱性

蔗糖: 蔗糖-1-果糖基转移酶（sucrose: sucrose 1-fructosyltransferase, 1-SST）以蔗糖为底物催化生成蔗果三糖等低聚合度的果聚糖.将从莴苣中克隆的1-SST基因重组到pCAMBIA1300-als中，构建了在CaMV 35S启动子调控下的植物表达载体，利用农杆菌介导的叶盘转化法将1-SST基因导入烟草中，PCR和Southern杂交检测表明获得了转基因植株，RT-PCR结果表明该基因在烟草中正常表达. 对T₀代转基因烟草进行的耐旱性分析结果表明，干旱胁迫6d的转基因植株丙二醛含量和电解质渗漏率显著低于未转基因对照，叶片相对含水量下降速度也明显比对照慢. 对转基因植株叶片糖分分析表明，转基因烟草植株积累果聚糖，并在干旱胁迫后含量明显增加，而未转基因对照植株不积累果聚糖. 在14%PEG溶液中未转基因烟草种子的萌发率仅为转基因烟草种子的一半；在附加200mmol/L甘露醇的培养基中未转基因烟草种子根的生长明显受到抑制，而转基因烟草根的生长发育正常. 以上研究结果表明，转1-SST基因烟草植株耐旱性的提高可能与该基因的表达有关.