Expression of a Thermostable Bacterial Cellulase in Transgenic Tobacco Plants

The bacterial gene of the thermostable endo--1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased bushiness and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.     

Morphology and Phytohormone Content in Transgenic Tobacco Plants Expressing Bacterial Thermostable Cellulase

Expression of the bacterial gene for thermostable -1,4-glucanase (cellulase) from Clostridium thermocellum in transgenic tobacco plants was shown to produce significant changes in tobacco plant structure and activities. The transgenic plants differed in their growth rate and morphology, and their hormonal status was affected. Thus, the transgenic plants expressing the gene for thermostable bacterial cellulase are a convenient model to study the role of -1,4-glucanases in plant physiological processes.     

Transgenic Tobacco plants expressing bacterial genes encoded the thermostable glucanases

It is shown that bacterial genes for thermostable beta-glucanases are expressed retaining their activity and substrate specificity. The leader peptide of the carrot extensin exerts effective secretion of the bacterial enzymes into the intercellular space of the plant tissue. Expression of the bacterial gene for beta-1,3-glucanase in plant tissues alters their morphogenetic potential. Regeneration of shoots from the calli of these plant lines requires a six- to eightfold increase in cytokinin (6-BAP) concentration in comparison with the control lines and the transgenic lines expressing beta-1,3-1,4-glucanase. Rooting of transgenic plants expressing the bacterial gene for beta-1,3-glucanase occurs much faster. The transgenic plants obtained in the study are proposed as model objects for investigating the role of glucanases in plants.     

Transgenic tobacco plants expressing the bacterial mc gene resist virus infection

A bacterial rnc gene coding for a double-stranded RNA-dependent RNase III endoribonuclease and a mutant, rnc70, were expressed in tobacco plants. The RNase III protein produced in the transgenic plants was the same size as the bacterial protein. Expression of the wild-type gene could cause stunting in some plant lines, but not in others. Expression of the mutant protein did not affect normal growth and development of the transgenic plants. Transgenic plants of the R1 and R2 generations, expressing the wild type, as well as a mutant protein, were resistant to infection by three disparate RNA plant viruses with a divided genome but not against two viruses with a single-stranded RNA genome. Introduction of the rnc gene in crop plants may provide resistance to economically important virus diseases.     

Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants

Selectable markers of bacterial origin such as the neomycin phosphotransferase type II gene, which can confer kanamycin resistance to transgenic plants, represent an invaluable tool for plant engineering. However, since all currently used antibiotic-resistance genes are of bacterial origin, there have been concerns about horizontal gene transfer from transgenic plants back to bacteria, which may result in antibiotic resistance. Here we characterize a plant gene, Atwbc19, the gene that encodes an Arabidopsis thaliana ATP binding cassette (ABC) transporter and confers antibiotic resistance to transgenic plants. The mechanism of resistance is novel, and the levels of resistance achieved are comparable to those attained through expression of bacterial antibiotic-resistance genes in transgenic tobacco using the CaMV 35S promoter. Because ABC transporters are endogenous to plants, the use of Atwbc19 as a selectable marker in transgenic plants may provide a practical alternative to current bacterial marker genes in terms of the risk for horizontal transfer of resistance genes.     

Ethylene formation and phenotypic analysis of transgenic tobacco plants expressing a bacterial ethylene-forming enzyme

A bacterial ethylene-forming enzyme (EFE) catalyzes oxygenation of 2-oxoglutarate to produce ethylene and carbon dioxide in contrast to a plant enzyme which uses 1-aminocyclopropane-1-carboxylic acid as a substrate. We constructed several lines of transgenic tobacco plants which expressed an EFE from Pseudomonas syringae pv. phaseolicola PK2. The gene encoding a chimeric protein consisting of EFE and beta-glucuronidase (GUS) was introduced into the tobacco genome using a binary vector which directs expression of the EFE-GUS fusion protein under the control of constitutive promoter of cauliflower mosaic virus 35S RNA. Two lines of transgenic plants produced ethylene at consistently higher rates than the untransformed plant, and their GUS activities were expressed in different tissues. A significant dwarf morphology observed in the transgenic tobacco displaying the highest ethylene production resembled the phenotype of a wild-type plant exposed to excess ethylene. These results demonstrate a potential use of bacterial EFE to supply ethylene as a hormonal signal via an alternative route using an ubiquitous substrate 2-oxoglutarate in plant tissues.     

Overexpression of poplar cellulase accelerates growth and disturbs the closing movements of leaves in sengon

In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant.     

Enhanced production of reducing sugars from transgenic rice expressing exo-glucanase under the control of a senescence-inducible promoter

We generated transgenic rice plants that express EXG1 exo-glucanase under the control of a senescence-inducible promoter. When a GUS coding sequence was connected to a promoter region of STAY GREEN (SGR) gene of rice and introduced into rice, GUS activity was specifically observed along with senescence. When an EXG1 cDNA was connected to the SGR promoter and introduced into rice, higher cellulase activities were detected after senescence. The EXG1 transgenic plants showed enhanced enzymatic saccharification efficiencies after senescence, but no significant difference of saccharification efficiencies was observed before senescence. The saccharification efficiencies were correlated with the cellulase activities in the transgenic plants. The EXG1 transgenic plants showed neither morphological abnormality nor sterility, both of which were observed when EXG1 was constitutively overexpressed. These results indicate that expression of cell wall degrading enzymes such as cellulase by a senescence-inducible promoter is one of the ways to enhance the saccharification ability of cellulosic biomass without affecting plant growth for efficient production of biofuels.     

Secondary metabolites in transgenic tobacco and potato: high accumulation of 4-hydroxybenzoic acid glucosides results from high expression of the bacterial gene ubiC

The bacterial gene ubiC encodes chorismate pyruvate-lyase (CPL), which converts chorismate to 4-hydroxybenzoate (4HB). The ubiC gene was expressed in tobacco (Nicotiana tabacum L., Solanaceae) and potato (Solanum tuberosum L., Solanaceae) under the control of the very strong constitutive plant promotor (ocs₃) mas. High accumulation of 4HB glucosides as new, artificial secondary metabolites was observed in the transgenic plants. 4HB glucoside content reached 5.1% of dry weight in tobacco cell cultures and 4.0% of dry weight in the leaves of potato shoots. This is the highest content of an artificial secondary metabolite produced by genetic engineering of plants reported so far. Surprisingly, no growth retardation and no phenotypical changes were observed in the transgenic cell cultures and plants. Glucosylation of 4HB was achieved by endogeneous, constitutively expressed glucosyltransferases. The total amount of 4HB glucoside acccumulated showed a strict linear dependence on the expression level of ubiC.     

大肠杆菌海藻糖合成酶基因对提高烟草抗逆性能的研究

编码大肠杆菌海藻糖合成酶的otsA基因由农杆菌介导引入野生型烟草植株并在花椰菜花叶病毒启动子序列 (CaMV35S)控制下获得表达。蒸发光散射高效液相层析法测定海藻糖实验表明 ,转基因烟草能够合成海藻糖 ,合成量达 1 4μg g叶片湿重 ;转基因烟草表现为耐盐性生长、干燥失重缓慢等抗逆表型。说明海藻糖合成酶otsA基因的引入 ,改变了烟草的糖代谢途径 ,同时也提高了植物的耐盐碱、耐干旱特性。     

新城疫病毒融合蛋白在转基因水稻叶片中的表达及其免疫原性

利用转基因植物作为生物反应器表达抗原蛋白具有广阔的应用前景。以新城疫病毒融合蛋白（NDVF）基因1.7kb全长编码区序列为外源基因与组成型表达的玉米泛素蛋白基因（Ubi）启动子和农杆菌胭脂碱合成酶基因（nos）终止子组成嵌合基因,构建了适宜于农杆菌介导转化水稻的转化载体pUNDV,经根癌农杆菌介导的遗传转化方法将由Ubi动子驱动的NDVF嵌合基因导入水稻细胞中,经潮霉素抗性筛选,共再生获得了6个独立的转基因株系。PCR分析结果表明NDVF基因已整合到水稻基因组中。ELISA和Western blot分析结果证实NDVF蛋白在部分转基因水稻叶片组织中获得表达,其中植株F5叶片组织中具有较高的表达水平。将F5叶片可溶性总蛋白皮下注射免疫BALB／c小鼠,结果表明能够诱导小鼠产生一定水平的NDVF蛋白特异抗体。     

Transgenic Tobacco Plants with a Fully Synthesized GFM CryIA Gene Provide Effective Tobacco Bollworm (Heliothis armigera) Control

GFM CrylA gene is a fully modified synthetic gene derived from insecticidal crystal prorein gene of Bacillus thuringiensis Berliner (Bt). It was synthesized based on the codon usage of plant genes instead of changing the primary sequences of amino acids of insecticidal crystal protein (ICP) gene of Bacillus thuringiensis Htibner. To test the function of the synthetic GFM CrylA gene, we introduced the GFM CrylA gene into tobacco plant cells via an Agrobacterium tumefacieus (Smith et Townsedn) Conn binary vector system. As expected, the GFM CrylA gene is expressed under control of the cauliflower mosaic virus (CaMV) 35S promoter and allows efficient production of lepidopteran insectspecific toxic proteins in the transformed tobacco plants. Bioassays using transgenic tobacco plants with tobacco bollworm showed that the transgenic tobacco plants expressing proteins of GFM CrylA gene had effective control to tobacco bollworm. In this paper the authors firstly report the complete synthesis of GFM CryIA gene and the construction of plant expression vector pGBI4AB. The authors performed introduction of the synthetic GFM CrylA gene into the tobacco plants, and the integration of GFM CrylA gene into tobacco genome was confirmed by Southern blot analysis of the tobacco genomic DNA. The gene was efficiently expressed in the transgenic tobacco plants and effective tobacco bollworm control was verified by the insect-bioassays.     

Construction of a phosphate transporter gene expression vector and its usage for tobacco transformation

A plant expression vector was constructed by inserting the phosphate transporter gene, LePT1, which was cloned from the tomato genome, into pCAMBIA2300. An agrobacterium-mediated system was used to transform tobacco and acquire transgenic plants. Analyses of the transgenic plants by PCR and RT-PCR indicated that the exogenous gene was integrated into and expressed by transgenic plants. The growth characteristics of T1 generation transgenic plants were examined, and the phosphate content of transgenic plants in a low-phosphate environment was found to be significantly higher than in wild-type plants.     

Tolerance of transgenic canola expressing 1-aminocyclopropane-1-carboxylic acid deaminase to growth inhibition by nickel.

Plant growth-promoting bacteria are useful to phytoremediation strategies in that they confer advantages to plants in contaminated soil. When plant growth-promoting bacteria contain the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, the bacterial cell acts as a sink for ACC, the immediate biosynthetic precursor of the plant growth regulator ethylene thereby lowering plant ethylene levels and decreasing the negative effects of various environmental stresses. In an effort to gain the advantages provided by bacterial ACC deaminase in the phytoremediation of metals from the environment two transgenic canola lines with the gene for this enzyme were generated and tested. In these transgenic canola plants, expression of the ACC deaminase gene is driven by either tandem constitutive cauliflower mosaic virus (CaMV) 35S promoters or the root specific rolD promoter from Agrobacterium rhizogenes. Following the growth of transgenic and non-transformed canola in nickel contaminated soil, it was observed that the rolD plants demonstrate significantly increased tolerance to nickel compared to the non-transformed control plants.     

Plant development inhibitory genes in binary vector backbone improve quality event efficiency in soybean transformation

Conventional Agrobacterium-mediated plant transformation often produces a significant frequency of transgenic events containing vector backbone sequence, which is generally undesirable for biotechnology applications. We tested methods to reduce the frequency of transgenic plants containing vector backbone by incorporating genes into the backbone that inhibit the development of transgenic plants. Four backbone frequency reduction genes, bacterial levansucrase (sacB), maize cytokinin oxidase (CKX), Phaseolus GA 2-oxidase (GA 2-ox), and bacterial phytoene synthase (crtB), each expressed by the enhanced CaMV 35S promoter, were placed individually in a binary vector backbone near the left border (LB) of binary vectors. In transformed soybean plants, the lowest frequency of backbone presence was observed when the constitutively expressed CKX gene was used, followed by crtB. Higher backbone frequencies were found among the plants transformed with the GA 2-oxidase and sacB vectors. In some events, transfer of short backbone fragments appeared to be caused by LB readthrough and termination within the backbone reduction gene. To determine the effect of the backbone genes on transformation frequency, the crtB and CKX vectors were then compared to a control vector in soybean transformation experiments. The results revealed that there was no significant transformation frequency difference between the crtB and control vectors, but the CKX vector showed a significant transformation frequency decrease. Molecular analysis revealed that the frequency of transgenic plants containing one or two copies of the transgene and free of backbone was significantly increased by both the CKX and crtB backbone reduction vectors, indicating that there may be a correlation between transgene copy number and backbone frequency.     

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of phytoremediation of polychlorinated biphenyls

The aim of this work was to construct transgenic plants with increased capabilities to degrade organic pollutants, such as polychlorinated biphenyls. The environmentally important gene of bacterial dioxygenase, the bphC gene, was chosen to clone into a plant of Nicotiana tabacum. The chosen bphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase, which cleaves the aromatic ring of dihydroxybiphenyl, and we cloned it in fusion with the gene for β-glucuronidase (GUS), luciferase (LUC) or with a histidine tail. Several genetic constructs were designed and prepared and the possible expression of desired proteins in tobacco plants was studied by transient expression. We used genetic constructs successfully expressing dioxygenase's genes we used for preparation of transgenic tobacco plants by agrobacterial infection. The presence of transgenic DNA , mRNA and protein was determined in parental and the first filial generation of transgenic plants with the bphC gene. Properties of prepared transgenic plants will be further studied.     

Seed-specific expression of a bacterial desensitized aspartate kinase increases the production of seed threonine and methionine in transgenic tobacco

In order to study the regulation of threonine and methionine synthesis in plant seeds, tobacco plants were transformed with a chimeric gene containing the coding DNA sequence of a mutant lysC gene from Escherichia coli fused to a promoter from a phaseolin seed storage protein gene. The bacterial mutant lysC gene codes for aspartate kinase (AK) which is desensitized to feedback inhibition by lysine and threonine. Increased AK activity, compared with control non-transformed plants, was detected in seeds but not in leaves, roots and flowers of the transgenic plants. This expression was accompanied by a significant increase in the levels of free threonine and methionine in the seed. The level of these amino acids also correlated positively with the levels of the bacterial enzyme. No alteration in plant phenotype and 'average seed weight' was observed in any of the transgenic plants, indicating that plant growth and seed development were normal. This study demonstrates, for the first time, that the threonine and methionine biosynthetic pathways are active in plant seeds. Thus, targeting of the production of favorable biosynthetic enzymes to plant seeds may represent a desirable molecular approach for production of crop plants with a more balanced nutritional quality.     

Transgenic Tobacco Plants Expressing Bacterial Genes for Thermostable Glucanases

It is shown that bacterial genes for thermostable -glucanases are expressed retaining their activity and substrate specificity. The leader peptide of the carrot extensin exerts effective secretion of the bacterial enzymes into the intercellular space of the plant tissue. Expression of the bacterial gene for -1,3-glucanase in plant tissues alters their morphogenetic potential. Regeneration of shoots from the calli of these plant lines requires a six- to eightfold increase in cytokinin (6-BAP) concentration in comparison with the control lines and the transgenic lines expressing -1,3-1,4-glucanase. Rooting of transgenic plants expressing the bacterial gene for -1,3-glucanase occurs much faster. The transgenic plants obtained in the study are proposed as model objects for investigating the role of glucanases in plants.