Construction of Synthetic Genes for Analogs of Spider Silk Spidroin 1 and Their Expression in Tobacco Plants

To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f9 coding for spidroin 1 analogs were 3"-fused in-frame with the reporter lichenase gene. The Tr2" weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.     

Experimental models of transgenic plants promising for innovative biotechnologies

Transgenic potato plants have been created that express recombinant proteins that are analogues of spidroin 1, the protein of the cobweb dragline. Expression of spidroin 1 hybrid genes possessing some repeated sequences is retained on going from model test tube-grown plants to crops. The expression level of the synthetic spidroin 1 genes and the level of accumulation of their products in plants depend on the type of promoter, number of repeats, organ specificity, and plant species, but not on the duration of plant material storage. The results show that the strategy based on construction and expression of hybrid proteins including a reporter protein makes it easier to select and analyze the expression of hybrid proteins in transgenic organisms.     

Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field production

Spider dragline silk is a unique biomaterial and represents nature's strongest known fibre. As it is almost as strong as many commercial synthetic fibres, it is suitable for use in many industrial and medical applications. The prerequisite for such a widespread use is the cost-effective production in sufficient quantities for commercial fibre manufacturing. Agricultural biotechnology and the production of recombinant dragline silk proteins in transgenic plants offer the potential for low-cost, large-scale production. The purpose of this work was to examine the feasibility of producing the two protein components of dragline silk (MaSp1 and MaSp2) from Nephila clavipes in transgenic tobacco. Two different promoters, the enhanced CaMV 35S promoter (Kay et al., 1987) and a new tobacco cryptic constitutive promoter, tCUP (Foster et al., 1999) were used, in conjunction with a plant secretory signal (PR1b), a translational enhancer (alfalfa mosaic virus, AMV) and an endoplasmic reticulum (ER) retention signal (KDEL), to express the MaSp1 and MaSp2 genes in the leaves of transgenic plants. Both genes expressed successfully and recombinant protein accumulated in transgenic plants grown in both greenhouse and field trials.     

Experimental models of transgenic plants promising for the modern technologies

Transgenic popato plants have been created which express recombinant proteins, analogues of spidroin 1, the protein of the cobweb skeleton thread. Expression of the hybrid spidroin 1 genes possessing some repeated sequences retains both in the model test-tube-growing plants and in the crops. Expression level of the synthetic spidroin 1 genes and the level of accumulation of their products in plants depend on the type of promoter, number of repeats, organ specificity and plant species but not on the duration of plant material storage. The results show that the strategy based on constuction and expression of hybrid proteins which include the reporter protein makes it easier to select and analyse expression of hybrid proteins in transgenic organisms.     

Purification of Spider Silk-elastin from Transgenic Plants and Application for Human Chondrocyte Proliferation

Research on spider silk proteins has led to the possibility of designing genetically engineered silks according to defined material properties. Here we show the efficient and stable production of spider silk-elastin fusion proteins in transgenic tobacco and potato plants by retention in the ER. The proteins were purified by a simple method, using heat treatment and 'inverse transition cycling'. Laboratory scale extraction of 1 kg tobacco leaf material leads to a yield of 80 mg pure recombinant spider silk-elastin protein. As a possible application, as well as to demonstrate biocompatibility, the growth of anchorage-dependent mammalian cells on spider silk-elastin coated culture plates was compared with conventional coatings such as collagen, fibronectin and poly-D-lysine. The anchorage-dependent chondrocytes showed similar growth behaviour and a rounded phenotype on collagen and on spider silk-elastin coated plates and the proliferation was remarkably superior to untreated polystyrene plates.     

Transgenic silkworms (<Emphasis Type="Italic">Bombyx mori</Emphasis>) produce recombinant spider dragline silk in cocoons

Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon. Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori.     

Acid extraction and purification of recombinant spider silk proteins

A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers.     

Production of spider silk proteins in tobacco and potato

Spider dragline silk is a proteinaceous fiber with remarkable mechanical properties that make it attractive for technical applications. Unfortunately, the material cannot be obtained in large quantities from spiders. We have therefore generated transgenic tobacco and potato plants that express remarkable amounts of recombinant Nephila clavipes dragline proteins. Using a gene synthesis approach, the recombinant proteins exhibit homologies of >90% compared to their native models. Here, we demonstrate the accumulation of recombinant silk proteins, which are encoded by synthetic genes of 420-3,600 base pairs, up to a level of at least 2% of total soluble protein in the endoplasmic reticulum (ER) of tobacco and potato leaves and potato tubers, respectively. Using the present expression system, spider silk proteins up to 100 kDa could be detected in plant tissues. When produced in plants, the recombinant spidroins exhibit extreme heat stability-a property that is used to purify the spidroins by a simple and efficient procedure.     

光敏启动子控制的酵母Hem1基因在烟草中表达

拟南芥hemA1基因启动子是一种光响应型启动子,它控制着植物体内5-氨基乙酰丙酸(ALA)昼夜节律型合成.将该启动子与酿酒酵母Hem1基因构建的二价载体转入烟草中,获得转基因植株.以转二价基因的烟草T0代种子为材料,用不同浓度卡那霉素(Km)溶液浸种,结果显示,种子发芽率和子叶绿化率随着Km浓度升高而降低.将1 000 mg·L-1Km溶液中长出的162株抗性植株移植至盆钵中培养,GUS检测出的阳性比率为92%.用特异引物PCR法检测Km抗性-GUS阳性植株,发现含有拟南芥HemA1基因启动子的比率为92.5%,含有酿酒酵母Hem1基因的比率为88%,含有二价重组基因的比率为84.2%.RT-PCR检测表明,Hem1基因在黑暗中的表达量明显低于光照下,证明光敏启动子有效地控制了结构基因Hem1的表达.生理指标分析表明,与野生型相比,转基因植株ALA合成速率、叶绿素含量明显增加,叶绿素b/a比值提高.野生型植株基部叶片SPAD值显著降低,而转基因植株基部叶片SPAD值保持较高水平.以上结果说明,外源二价基因已经成功整合到烟草基因组中,并且能够在光照条件下过量表达,合成过量ALA.     

Enhanced resistance to phytopathogenic bacteria in transgenic tobacco plants with synthetic gene of antimicrobial peptide cecropin P1

Plasmids with a synthetic gene of the mammalian antimicrobial peptide cecropin P1 (cecP1) controlled by the constitutive promoter 35S RNA of cauliflower mosaic virus were constructed. Agrobacterial transformation of tobacco plants was conducted using the obtained recombinant binary vector. The presence of gene cecP1 in the plant genome was confirmed by PCR. The expression of gene cecP1 in transgenic plants was shown by Northern blot analysis. The obtained transgenic plants exhibit enhanced resistance to phytopathogenic bacteria Pseudomonas syringae, P. marginata, and Erwinia carotovora. The ability of transgenic plants to express cecropin P1 was transmitted to the progeny. F1 and F2 plants had the normal phenotype (except for a changed coloration of flowers) and retained the ability to produce normal viable seeds upon self-pollination. Lines of F1 plants with Mendelian segregation of transgenic traits were selected.     

六棱大麦HVA1基因在烟草中遗传转化的研究

本研究依据HVA1基因序列克隆六棱大麦HVA1基因cDNA片段,构建Ubiquitin启动子驱动下的植物表达载体pCAMBIA1300-HVA1。然后通过三亲杂交法将重组质粒PCAMBIA1300-HVA1转入农杆菌LBA4404,并采用农杆菌介导法转化烟草。经PCR,PCR-Southern blotting和RT-PCR检测表明HVA1基因已整合进烟草基因组,并在转录水平上获得表达。功能验证的结果显示,转基因植株叶片的保水率提高了近1倍,暗示转基因烟草具有一定的抗旱潜力。     

Functional Characterization of the Tau Class Glutathione-S-Transferases Gene (SbGSTU) Promoter of Salicornia brachiata under Salinity and Osmotic Stress

Reactive oxygen or nitrogen species are generated in the plant cell during the extreme stress condition, which produces toxic compounds after reacting with the organic molecules. The glutathione-S-transferase (GST) enzymes play a significant role to detoxify these toxins and help in excretion or sequestration of them. In the present study, we have cloned 1023 bp long promoter region of tau class GST from an extreme halophyte Salicornia brachiata and functionally characterized using the transgenic approach in tobacco. Computational analysis revealed the presence of abiotic stress responsive cis-elements like ABRE, MYB, MYC, GATA, GT1 etc., phytohormones, pathogen and wound responsive motifs. Three 5’-deletion constructs of 730 (GP2), 509 (GP3) and 348 bp (GP4) were made from 1023 (GP1) promoter fragment and used for tobacco transformation. The single event transgenic plants showed notable GUS reporter protein expression in the leaf tissues of control as well as treated plants. The expression level of the GUS gradually decreases from GP1 to GP4 in leaf tissues, whereas the highest level of expression was detected with the GP2 construct in root and stem under control condition. The GUS expression was found higher in leaves and stems of salinity or osmotic stress treated transgenic plants than that of the control plants, but, lower in roots. An efficient expression level of GUS in transgenic plants suggests that this promoter can be used for both constitutive as well as stress inducible expression of gene(s). And this property, make it as a potential candidate to be used as an alternative promoter for crop genetic engineering.     

抗草甘膦抗虫植物表达载体的构建及其转基因烟草的分析

构建了含草甘膦抗性突变基因（aroAM12）和人工合成重组Bt抗虫基因（Bts1m）的植物表达载体pCM12_s1m。aroAM12基因的表达由CaMV35S启动子控制,Bts1m基因的表达由2E_CaMV35S启动子和Ω因子控制。通过农杆菌介导,将aroAM12和Bts1m基因转化到烟草中,转基因烟草通过在含草甘膦的MS培养基上筛选而获得。Southern blot分析表明所有经过草甘膦筛选出的转化植株都整合有aroAM12基因,约70%的转化植株同时整合有aroAM12和Bts1m基因。Northern blot、Immunodot blot分析进一步证明整合的两个基因在转录、翻译水平上均进行了表达,不同植株之间表达存在着差异。草甘膦抗性和虫试实验证明,获得的转基因烟草对草甘膦和烟青虫具有很强的抗性。     

Containment of Extended Length Polymorphisms in Silk Proteins

The spider silk gene family to the current date has been developed by gene duplication and homogenization events as well as conservation of crucial sequence parts. These evolutionary processes have created an amazing diversity of silk types each associated with specific properties and functions. In addition, they have led to allelic and gene variants within a species as exemplified by the major ampullate spidroin 1 gene of Nephila clavipes. Due to limited numbers of individuals screened to date little is known about the extent of these heterogeneities and how they are finally manifested in the proteins. Using expanded sample sizes, we show that sequence variations expressed as deletions or insertions of tri-nucleotides lead to different sized and structured repetitive units throughout a silk protein. Moreover, major ampullate spidroins 1 can quite dramatically differ in their overall lengths; however, extreme variants do not spread widely in a spider population. This suggests that a certain size range stabilized by purifying selection is important for spidroin 1 gene integrity and protein function. More than one locus for spidroin 1 genes possibly exist within one individual genome, which are homogenized in size, are differentially expressed and give a spider a certain degree of adaptation on silk’s composition and properties. Such mechanisms are shared to a lesser extent by the second major ampullate spidroin gene.     

Seed-transmissible expression of mammalian metallothionein in transgenic tobacco

A binary plasmid was constructed to contain the mouse metallothionein c-DNA, the constitutive 35S promoter from cauliflower mosaic virus, the polyadenylation signal from the pea rbcS-E9 gene and several selectable markers. The plasmid was transferred to Agrobacterium tumefaciens and the leaf disc method was used to transform tobacco. Callus and shoots were regenerated in the presence of kanamycin and transformed plants were obtained. Southern, Northern and Western blot analysis demonstrated integration and expression of the metallothionein gene in transformed callus and transgenic plants. The gene is transmitted to and expressed in seed derived progeny as a dominant Mendelian trait.     

Morphological features of the transgenic tobacco plant shoot expressing the 3-hydroxy-3-methylglutagyl-CoA reductase (<Emphasis Type="Italic">HMG1</Emphasis>) gene in the direct and reverse orientations towards the promoter

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG1) catalyzes the formation of mevalonic acid, the key intermediate of the cytosolic isoprenoid synthesis pathway. The parameters of stem and leaf growth were studied in the transgenic tobacco plants that express the HMG1 gene in both sense and antisense orientations towards the constitutive promoter. The transgenic plant height did not significantly differ from that of the control plants, though the plants carrying the sense copy of the HMG1 gene were considerably taller than plants that carried the antisense gene copy. Plants carrying an extra copy of the HMG1 gene were also characterized by increased leaf area. The number of mesophyll cells calculated per square unit of transgenic plants leaves was smaller than in the control plant leaves, though their volume was not considerably changed in any of the variants, suggesting changes in the cell packing density in leaves.     

Enhancing plant growth and biomass production by overexpression of GA20ox gene under control of a root preferential promoter

Overexpression of GA20 oxidase gene has been a recent trend for improving plant growth and biomass. Constitutive expression of GA20ox has successfully improved plant growth and biomass in several plant species. However, the constitutive expression of this gene causes side-effects, such as reduced leaf size and stem diameter, etc. To avoid these effects, we identified and employed different tissue-specific promoters for GA20ox overexpression. In this study, we examined the utility of At1g promoter to drive the expression of GUS (β-glucuronidase) reporter and AtGA20ox genes in tobacco and Melia azedarach. Histochemical GUS assays and quantitative real-time-PCR results in tobacco showed that At1g was a root-preferential promoter whose expression was particularly strong in root tips. The ectopic expression of AtGA20ox gene under the control of At1g promoter showed improved plant growth and biomass of both tobacco and M. azedarach transgenic plants. Stem length as well as stem and root fresh weight increased by up to 1.5–3 folds in transgenic tobacco and 2 folds in transgenic M. azedarach. Both tobacco and M. azedarach transgenic plants showed increases in root xylem width with xylem to phloem ratio over 150–200% as compared to WT plants. Importantly, no significant difference in leaf shape and size was observed between At1g::AtGA20ox transgenic and WT plants. These results demonstrate the great utility of At1g promoter, when driving AtGA20ox gene, for growth and biomass improvements in woody plants and potentially some other plant species.

     

Over-expression of Osmotin Induces Proline Accumulation and Confers Tolerance to Osmotic Stress in Transgenic Tobacco

Osmotin has been implicated in conferring tolerance to drought and salt stress in plants. We have over-expressed the osmotin gene under the control of constitutive CaMV 35S promoter in transgenic tobacco, and studied involvement of the protein in imparting tolerance to salinity and drought stress. The transgenic plants exhibited retarded leaf senescence and improved germination on a medium containing 200mM NaCl. Further, the transgenics maintained higher leaf relative water content (RWC), leaf photosynthesis and free proline content than the wild type plants during water stress and after recovery from stress. When subjected to salt stress (200mM NaCl), the transgenic plants accumulated significantly more proline than the wild type plants. These results suggest the involvement of the osmotin-induced increase in proline in imparting tolerance to salinity and drought stress in transgenic plants over-expressing the osmotin gene.