Overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica acquires resistance to salt stress in transgenic tobacco plants

To test the role of the heat shock protein DnaK/Hsp70 in salt tolerance, transgenic plants of Nicotina tabacum cv Petit Havana SR1 were made with DnaK1 from a halotolerant cyanobacterium Aphanothece halophytica (A. halophytica) overexpressed in the cytosol. The growth rate and photosynthetic activities of the transgenic and control tobacco plants were similar under non-stressed conditions. The CO₂ assimilation rate of the control plants decreased with increasing concentration of NaCl. After 3 days of treatment with 0.6 M NaCl, the CO₂ fixation rate decreased to 40% of that in the non-stressed plants whereas its activity in the transgenic plants was about 85% of that in the non-stressed plants. Similar results were observed for the stomatal transpiration. The sodium contents in leaves of the control plants were significantly increased by salt stress whereas those in the transgenic plants remained at levels similar to those in the non-stressed plants. Total protein contents and ribulose 1,5-bis phosphate carboxygenase and oxygenase (RuBisCO) levels were decreased by salt stress in both the transgenic and control plants but the decrease was slight in the transgenic tobacco. All these data clearly indicate that the expression of DnaK1 from a halotolerant cyanobacterium A. halophytica improved the salt tolerance of the tobacco plant.     

Cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum and transgenic CaPGIP1 in tobacco in relation to increased resistance to two fungal pathogens

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit fungal endopolygalacturonases (PGs). The PGIPs directly reduce the aggressive potential of PGs. Here, we isolated and functionally characterized three members of the pepper (Capsicum annuum) PGIP gene family. Each was up-regulated at a different time following stimulation of the pepper leaves by Phytophthora capcisi and abiotic stresses including salicylic acid, methyl jasmonate, abscisic acid, wounding and cold treatment. Purified recombinant proteins individually inhibited activity of PGs produced by Alternaria alternata and Colletotrichum nicotianae, respectively, and virus-induced gene silencing in pepper conferred enhanced susceptibility to P. capsici. Because three PGIP genes acted similarily in conferring resistance to infection by P. capsici, and because individually purified proteins showed consistent inhibition against PG activity of both pathogens, CaPGIP1 was selected for manipulating transgenic tobacco. The crude proteins from transgenic tobacco exhibited distinct enhanced resistance to PG activity of both fungi. Moreover, the transgenic tobacco showed effective resistance to infection and a significant reduction in the number of infection sites, number of lesions and average size of lesions in the leaves. All results suggest that CaPGIPs may be involved in plant defense response and play an important role in a plant’s resistance to disease.     

Characterization of a new antifungal lipid transfer protein from wheat

Lipid transfer proteins (LTPs) are members of the family of pathogenesis-related proteins (PR-14) that are believed to be involved in plant defense responses. In this study, a novel gene Ltp 3F1 encoding an antifungal protein from wheat (Sumai 3) was subcloned, overexpressed in Escherichia coli BL-21 (DE3) and enriched using ammonium sulfate fractionation followed by gel permeation chromatography. Molecular phylogeny analyses of wheat Ltp 3F1 gene showed a strong identity to other plant LTPs. Predicted three-dimensional structural model showed the presence of 6 α-helices and 9 loop turns. The active site catalytic residues Gly30, Pro50, Ala52 and Cys55 may be suggested for catalyzing the reaction involved in lipid binding. SDS–PAGE analysis confirmed the production of recombinant fusion protein. The LTP fusion protein exhibited a broad-spectrum antifungal activity against Alternaria sp., Rhizoctonia solani, Curvularia lunata, Bipolaris oryzae, Cylindrocladium scoparium, Botrytis cinerea and Sarocladium oryzae. Gene cassette with cyanamide hydratase (cah) marker and Ltp 3F1 gene was constructed for genetic transformation in tobacco. Efficient regeneration was achieved in selective media amended with cyanamide. Transgenic plants with normal phenotype were obtained. Results of PCR and Southern, Northern and Western hybridization analyses confirmed the integration and expression of genes in transgenic plants. Experiments with detached leaves from transgenic tobacco expressing Ltp 3F1 gene showed fungal resistance. Due to the innate potential of broad-spectrum antifungal activity, wheat Ltp 3F1 gene can be used to enhance resistance against fungi in crop plants.     

Modulation of flowering responses in different Nicotiana varieties

We have identified and characterized a FLOWERING PROMOTING FACTOR 1(FPF1) gene from tobacco (NtFPF1). Over-expression of NtFPF1 leads to early flowering in the day-neutral tobacco Nicotiana tabacum cv. Hicks, and under inductive photoperiods also in the short-day Nicotiana tabacum cv. Hicks Maryland Mammoth (MM) tobacco and the long-day plant Nicotiana sylvestris. N. sylvestris wild-type plants remained in the rosette stage and never flowered under non-inductive short-days, whereas 35S::NtFPF1 transgenic plants bolted but did not flower. However, if treated with gibberellins, transgenic N. sylvestrisplants flowered much faster under non-inductive short days than corresponding wild type plants, indicating an additive effect of gibberellins and the NtFPF1 protein in flowering time control. The day-neutral wild type cv. Hicks and the short-day cv. Hicks MM plants exhibit an initial rosette stage, both under short- and long-days. In the transgenic lines, this rosette stage was completely abolished. Wild-type plants of cv. Hicks MM never flowered under long days; however, all transgenic lines over-expressing NtFPF1 flowered under this otherwise non-inductive photoperiod.     

Characterization of the GLP13 gene promoter in Arabidopsis thaliana

In transgenic plants, for many applications it is important that the inserted genes are expressed in a tissue-specific manner. This in turn could help better understanding their roles in plant development. Germin-like proteins (GLPs) play diverse roles in plant development and defense responses. In order to understand the functions and regulation of the GLP13 gene, its promoter (762 bp) was cloned and fused with a β-glucuronidase (GUS) reporter gene for transient expression in Arabidopsis thaliana and tobacco (Nicotiana tabacum cv. K326). Histochemical analysis of the transgenic plants showed that GUS was specifically expressed in vascular bundles predominantly in phloem tissue of all organs in Arabidopsis. Further analyses in transgenic tobacco also identified similar GUS expression in the vascular bundles.     

Development of analytical tools for evaluating the effect of T-DNA chimeric integration on transgene expression in vegetatively propagated plants

T-DNA chimeric integration and unexpected transgene expression are relevant constraints affecting transgenic plants. This study aims to properly investigate the occurrence of these events and to what extent they may be related. The final goal is to develop an effective screening tool for earlier selection of proper transgenic lines. A strategy based on qPCR and Southern blot was adopted for evaluating gus and Egfp chimerism degree in transgenic Vitis vinifera cv ‘Chardonnay’. Of nine transgenic lines, one had a very high chimerism value, which was shown to be associated with minimal transgene expression. The evaluation of the gus gene over time and space on a line selected as a model showed that transgene’s chimerism was stable and uniform throughout plant tissues whilst its expression was highly variable. Transgene chimerism issue was investigated in detail and useful hints were given for selecting the most favorable transgenic plants and for proper planning of in vitro and ex vitro experiments.     

Overexpression of MsALMT1, from the Aluminum-Sensitive Medicago sativa, Enhances Malate Exudation and Aluminum Resistance in Tobacco

Aluminum (Al) toxicity is a major limiting factor for plant growth and crop production in acidic soils. Al-induced organic acid (OA) exudation plays an important role in plant Al resistance. The exudation of OAs is mediated by membrane-localized OA transporters. In our previous study, a gene encoding the Al-induced malate transporter (MsALMT1) was identified in the roots of the Al-sensitive plant Medicago sativa L. cv. Yumu no. 1 (YM1). To further validate the function of MsALMT1, transgenic plants that overexpressed MsALMT1 under the control of the CaMV 35S (35S) promoter were generated. This transgenic tobacco showed an enhanced capacity for malate efflux and better Al resistance than wild type (WT) plants after exposure to 30 μM Al for 24 h. The Al content in the transgenic plant roots decreased to 40–52 % of that in WT plant roots. These results demonstrate that MsALMT1 is an Al-resistant gene in YM1 and encodes a malate transporter, the overexpression of which effectively enhances the Al resistance of transgenic tobacco plants.     

小麦盐华南象草PpCAD基因的亚细胞定位及其在转基因烟草中的异源表达迫相关基因的克隆与表达分析

该研究根据已克隆的华南象草(Pennisetum purpureum cv.Huanan)肉桂醇脱氢酶(CAD)基因PpCAD的cDNA序列,构建亚细胞定位载体pAN580-PpCAD,用PEG介导法转化象草原生质体,以探究PpCAD蛋白在细胞内的定位;同时构建植物过表达载体pBA002-PpCAD,通过农杆菌介导法在烟草中异源表达,以研究PpCAD基因与植物木质素合成的关系。结果显示:(1)PpCAD定位在象草原生质体的细胞质内;(2)过表达载体pBA002-PpCAD转化烟草后获得27株转基因烟草,其中25株PCR鉴定为阳性;(3)半定量RT-PCR检测6株转基因烟草后发现,PpCAD基因在不同植株的表达量存在差异,通过Southern杂交检测后发现该差异与目的基因插入的拷贝数有关;(4)6株转基因烟草和野生型烟草表型上没有明显差异,除目的基因多拷贝插入的植株OEC6外,木质素含量有不同程度的提高,最高比野生型提高了56.50%。研究表明,PpCAD是一个细胞质蛋白,在烟草中过表达PpCAD能够提高植株木质素含量,表明PpCAD基因参与了植物的木质素合成,可用于象草的木质素调控研究。     

Induced expression of <Emphasis Type="Italic">Serratia marcescens</Emphasis> ribonuclease III gene in transgenic <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. cv. SR1 tobacco plants

Transgenic Nicotiana tabacum L. cv. SR1 plants, characterized by an increase in the level of dsRNA-specific hydrolytic activity after induction by wounding, were obtained. The Solanum lycopersicum anionic peroxidase gene promoter (new for plant genetic engineering) was for the first time used for the induced expression of the target Serratia marcescens RNase III gene. Upon infection with the tobacco mosaic virus (TMV), the transgenic plants of the obtained lines did not differ significantly from the control group in the level of TMV capsid protein accumulation. In general, no delay in the development of the infection symptoms was observed in transgenic plants as compared with the control group. The obtained transgenic plants represent a new model for the study of the biological role of endoribonucleases from the RNase III family, including in molecular mechanisms of resistance to pathogens.     

Some Physiological Parameters and Seed Lipid Composition of Transgenic Tobacco Plant

To see the effects of foreign gene introduction on the physiological performance and the quality and quantity of seed lipids, we studied transgenic tobacco plant as a model system, as tobacco seeds are oil seeds. Using Agrobacterium Ti plasmid based vectors, tobacco plants cv Petit Havana were transformed by NPT II gene as selectable marker. Transformed T₀ generation plants raised in tissue culture were transferred to pots and selfed. From the seeds, T₁ generation plants were grown in pots and their physiological performance was assessed. The transgenic plants showed slightly slower rates of germination and growth. Total chlorophyll content, chlorophyll a/b ratio and specific leaf weight, however, remained unchanged. The transgenic plants also had delayed flowering. However, total protein, lipid content and fatty acid composition of lipids of seeds in transgenic plants did not show appreciable difference from the seeds from control plants. Thus the physiological cost of transgenic plant for the extra genetic load was only marginal, if any.     

Physiological and proteomic analysis of plant growth enhancement by the rhizobacteria <Emphasis Type="Italic">Bacillus</Emphasis> sp. JS

In this study, the effects of the plant growth-promoting rhizobacterium (PGPR), Bacillus sp. JS on the growth of tobacco (Nicotiana tabacum ‘Xanthi’) and lettuce (Lactuca sativa ‘Crispa’), were evaluated by comparing various growth parameters between plants treated with the bacterium and those exposed to water or nutrient broth as control. In both tobacco and lettuce, fresh weight and length of shoots were increased upon exposure to Bacillus sp. JS. To explain the overall de novo expression of plant proteins by bacterial volatiles, two-dimensional gel electrophoresis was performed on samples from PGPR-treated tobacco plants. Our results showed that chlorophyll a/b binding proteins were significantly up-regulated, and total chlorophyll content was also increased. Our findings indicate the potential benefits of using Bacillus sp. JS as a growth-promoting factor in agricultural practice, and highlight the need for further research to explore these benefits.