Cloning of Helicobacter pylori urease subunit B gene and its expression in tobacco (Nicotiana tabacum L.)

Vaccines produced by transgenic plants would have the potential to change the traditional means of production and inoculation of vaccines, and to reduce the cost of vaccine production. In the present study, an UreB antigen gene from a new Helicobacter pylori strain ZJC02 was cloned into the binary vector pBI121 which contains a CaMV35S promoter and a kanamycin resistance gene, and then transformed UreB into tobacco leaf-disc by Agrobacterium-mediated method. A total of 50 regenerated plants with kanamycin resistance were obtained in the selection media. The 35 putative transgenic individuals were tested and verified the presence and integration of the UreB into the nuclear genome of tobacco plants by PCR, PCR-southern, and Southern analyses. Expression of UreB gene in the tobacco plants was confirmed by RT-PCR and Western Blot analysis using polyclonal human antiserum. To our knowledge, this is the first report of the expression of Helicobacter pylori UreB antigen gene in a plant system, suggesting a major step in the production of plant-based vaccines for Helicobacter pylori.     

转基因番木瓜作为抗结核植物口服疫苗的初步研究

转基因植物疫苗的研制,可改变传统的疫苗接种方式和接种手段,大大降低疫苗的生产成本,具有广阔的应用前景。结核病的泛滥使抗结核的免疫预防急需开发新型的疫苗,研制抗结核的转基因植物疫苗具有重大的理论和实践意义。本文对具有良好免疫原性的结核杆菌分泌蛋白ESAT—6基因,进行了修饰改造,构建了带潮霉素选择抗性基因的植物表达载体和根癌农杆菌工程菌;采用叶盘法转化热带水果番木瓜,获得了13株抗性植株,通过PCR、Southern blot和RNA dot blot分子检测,确认了4株为转基因植株。番木瓜ESAT—6转基因植株的获得,为进一步的结核病植物口服疫苗的免疫研究和新型抗结核疫苗的研制与开发奠定了基础。     

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.     

Transformation of Populus tomentosa with Insecticidal Cowpea Proteinase Inhibitor Gene

Cowpea trypsin inhibitor (CpTI) gene, an insecticidal gene, was introduced into poplar ( Populus tomentosa Carr. ) by gene transformation mediated by Agrobacterium tumefac/ens (Smith et Townsend) Conn. The influences on regeneration and transformation frequency of poplar by the concentration and addition of kanamycin were compared. Kanamycin resistant (Kmr) plantlets were obtained by 3 -4 cycles screening in selective condition. The ability of leaf regeneration and shoot subculture and rooting from the transformed and non-transformed plants in the presence of 50 mg/L kanamycin was examined. The presence of CpTI gene in the transgenic plants were confirmed by PCR and PCR-Southern blot. Assay on proteinase inhibition activity demonstrated that leaf protein extracts of the transgenic poplar showed higher inhibition activity against trypsin than that of control plants.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.) using root explants

An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed. Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calli were induced in media containing 0.1 mg l(-1) thidiazuron (TDZ), 3.0 mg l(-1) N(6)-benzylaminopurine, 100 mg l(-1) kanamycin and 500 mg l(-1) cefotaxime. The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg l(-1) kanamycin sulphate. Transgenic plants were raised in pots and seeds subsequently collected from mature fruits. Histochemical GUS assay and polymerase chain reaction analysis of field-established transgenic plants and their offsprings confirmed the presence of the GUS and NPTII genes, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both the NPTII and GUS genes.     

Transformation of Brassica napus with Agrobacterium tumefaciens based vectors

A reproducible system to produce transgenic Brassica napus plants has been developed using stem segments. Stem segments from 6–7 week old plants were inoculated with an Agrobacterium tumefaciens strain containing a disarmed tumor-inducing plasmid pTiT37-SE carrying a chimeric bacterial gene encoding kanamycin resistance (pMON200). Stem explants were cocultured for 2 days before transfer to kanamycin selection medium. Shoots regenerated directly from the explant in 3–6 weeks and were excised, dipped in Rootone®, and rooted in soil. Transformation was confirmed by opine production, kanamycin resistance, and DNA blot hybridization in the primary transformants. Final proof of transformation was demonstrated by the co-transfer of opine production and kanamycin resistance to progeny in a Mendelian fashion. Over 200 transgenic Brassica napus plants have been produced using this system.Abbreviations BA 6-benzyladenine - NAA -naphthalene-acetic acid - T-DNA transferred DNA into plants - IBA indole butyric acid - IAA indole acetic acid - TXD Tobacco Xanthi diploid suspension cells     

苜蓿花叶病毒外壳蛋白基因对红三叶的遗传转化及转基因植株的抗病性分析

以红三叶(Trifolium pratense L．)子叶为转化体,用农杆菌介导法将外源的苜蓿花叶病毒外壳蛋白基因AMV4转入红三叶,经过筛选、分化和再生,得到了具有卡拉霉素抗性的转基因植株。对这些植株进行PCR、Southern印迹杂交和Northern杂交分析,结果表明,外源目的基因已经整合到红三叶基因组中并且得到了表达。对Northern分析呈阳性的植株进行了抗病性检测,结果表明,表达苜蓿花叶病毒外壳蛋白基因的植株病症减轻,发病率、病情指数及病毒积累量都明显低于对照,有的甚至不表现症状,达到了免疫的程度。     

轮状病毒外壳蛋白VP7在转基因番茄果实中的特异表达

将轮状病毒外壳蛋白VP7基因克隆到含有番茄果实特异性启动子TFP的植物表达载体pTF ,并转化到根癌农杆菌 (Agrobacteriumtumefaciens)菌株EHA10 5中 ,采用叶盘转化法转化番茄 (LycopersiconesculentumMill.)栽培品种TX0 0 14 ,获得了转基因植株。经PCR、PCR Southernblot和Southernblot分析表明VP7基因已整合到转基因番茄植株的核基因组中 ,RT PCR、Westernblot结果表明VP7蛋白在果实中获得了特异表达     

Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus

In vitro regeneration and biolistic transformation procedures were developed for several commercial chrysanthemum Dendranthema grandiflora Tzvelev, syn. Chrysanthemum morifolium Ramat. cultivars using leaf and stem explants. Studies on the effect of several growth regulators and kanamycin on chrysanthemum regeneration were conducted, and a step-wise procedure to optimize kanamycin selection and recovery of transgenic plants was developed. A population of putative transformed chrysanthemum plants cvs. Blush, Dark Bronze Charm, Iridon, and Tara, was obtained after bombardment with tungsten microprojectiles coated with the binary plasmid pBIN19 containing the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) and the marker gene neomycin phosphotransferase (NPT II). PCR analysis of 82 putative transgenic plants selected on kanamycin indicated that the majority of the lines (89%) were transformed and contained both genes (71%). However, some transgenic lines contained only one of the genes: either the NPT II (15%) or the TSWV (N) gene (14%). Southern blot analysis on selected transgenic lines confirmed the integration of the TSWV (N) gene into the chrysanthemum genome. These results demonstrate the development of an efficient procedure to transfer genetic material into the chrysanthemum genome and selectively regenerate transgenic chrysanthemum plants at frequencies higher than previously reported.     

Insect Resistance of Transformed Tobacco Plants with Gene of the Spider Insecticidal Peptide

Tobacco (Nicotiana tabacum)leaves were transformed with Agrobacterium tumefaciens LBA4404 containing the insecticidal peptide gene. Thirty regenerated kanamycin resistant plants were obtained, among which three showed stronger toxicity to Heliothis armigera by feeding experiments. In comparison with feeding of the control plants, mortality of the insects fed on transgenic plants was significantly higher and the growth of the survived insects was remarkably retarded. Results of PCR Southern blot and Northern blot showed that insecticidal peptide gene has been transferred into the genome of these three plants and expressed efficiently to confer the insect resistance of the transgenic plants.     

Survival and escape of Agrobacterium tumefaciens in triploid hybrid lines of Chinese white poplar transformed with two insect-resistant genes

Two partly modified insect-resistant genes (BtCryI Ac gene [Bt gene toxin against Lepidopterean insects] and API gene [arrowhead proteinase inhibitor]) were transferred to the triploid hybrid of Chinese white poplar ((Populus tomentosa Carr. × Populus bolleana Louche) × Populus tomentosa Carr.) mediated by A. tumefaciens. The survival of Agrobacterium in transgenic plants was examined during the processes of transplanting and subculturing on the nutrient medium. The results suggested that 80% of the plants, which were obtained by repeated selection on media added with 50 mg/L kanamycin and 300 mg/l carbenicillin, showed positive reactions after examination using molecular methods. The ELISA test indicated that the Bt toxoprotein was expressed in seven of the transgenic sub-clones. Leaves, stems, and roots of all the 28 transgenic plants were cultured on the YEB medium added with 50 mg/L kanamycin, and it was found that Agrobacterium survived in three sub-clones (33, 37, 5) and could have existed for 24 months in the bottle. These three transgenic sub-clones were transplanted and cultivated for one month in the room, and then the target Agrobacterium was found in rhizosphere of the sub-clone 33.     

Transgenic loblolly pine (Pinus taeda L.) plants expressing a modified delta-endotoxin gene of Bacillus thuringiensis with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud

A synthetic version of the CRY1Ac gene of Bacillus thuringiensis has been used for the transformation of loblolly pine (Pinus taeda L.) using particle bombardment. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Expression vector pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) CRY1Ac coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator sequences, and the neomycin phosphotransferase II (NPTII) gene controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected on media with kanamycin. Shoot regeneration was induced from the kanamycin-resistant calli, and transgenic plantlets were then produced. More than 60 transformed plants from independent transformation events were obtained for each loblolly pine genotype tested. The integration and expression of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern hybridization, by Northern blot analysis, and by Western blot analysis. Effective resistance of transgenic plants against Dendrolimus punctatus Walker and Crypyothelea formosicola Staud was verified in feeding bioassays with the insects. The transgenic plants recovered could represent a good opportunity to analyse the impact of genetic engineering of pine for sustainable resistance to pests using a B. thuringiensis insecticidal protein. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform.     

导入蜘蛛杀虫肽基因的烟草具有抗虫性

用带有杀虫肽基因的农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍ ｅｆａｃｉｅｎｓ）ＬＢＡ４４０４ 转化烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ）叶片,共获得３０ 株抗卡那霉素的再生植株． 用这些再生植株对棉铃虫（Ｈｅｌｉｏｔｈｉｓａｒｍ ｉｇｅｒａ）进行毒力测定,有３ 株转杀虫肽基因植株对棉铃虫有较强抗性． 与对照相比,这３ 株转基因烟草的杀虫率可达３０％～４５％ ,并能显著抑制昆虫蜕皮和生长发育,表现出明显的抗虫作用． 以这３ 株为主进行了ＰＣＲ 扩增及Ｎｏｒｔｈｅｒｎ ｂｌｏｔ实验,结果表明杀虫肽基因已插入到这３ 株植株的基因组中并表达出有活性的杀虫肽     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants

To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV 16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV 16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.