Engineered tobacco and microalgae secreting the fungal laccase POXA1b reduce phenol content in olive oil mill wastewater

Olive oil mill wastewaters (OMWs) are characterised by low pH and a high content of mono- and polyaromatic compounds that exert microbial and phytotoxic activity. The laccase cDNA of the poxA1b gene from Pleurotus ostreatus, carrying a signal peptide sequence for enzyme secretion and driven by the CaMV 35S promoter, was cloned into a plant expression vector. Nuclear genetic transformation was carried out by co-cultivation of Agrobacterium tumefaciens with tobacco cv Samsun NN leaves and cells of five different microalgae accessions belonging to the genera Chlamydomonas, Chlorella and Ankistrodesmus. Transgenic plants and microalgae were able to express and secrete the recombinant laccase in the root exudates and the culture medium, respectively. In comparison to untransformed controls, the ability to reduce phenol content in OMW solution was enhanced up to 2.8-fold in transgenic tobacco lines and by up to about 40% in two microalgae accessions. The present work provides new evidence for metabolic improvement of green organisms through the transgenic approach to remediation.     

Engineered tobacco and microalgae secreting the fungal laccase POXA1b reduce phenol content in olive oil mill wastewater

Olive oil mill wastewaters (OMWs) are characterised by low pH and a high content of mono- and polyaromatic compounds that exert microbial and phytotoxic activity. The laccase cDNA of the poxA1b gene from Pleurotus ostreatus, carrying a signal peptide sequence for enzyme secretion and driven by the CaMV 35S promoter, was cloned into a plant expression vector. Nuclear genetic transformation was carried out by co-cultivation of Agrobacterium tumefaciens with tobacco cv Samsun NN leaves and cells of five different microalgae accessions belonging to the genera Chlamydomonas, Chlorella and Ankistrodesmus. Transgenic plants and microalgae were able to express and secrete the recombinant laccase in the root exudates and the culture medium, respectively. In comparison to untransformed controls, the ability to reduce phenol content in OMW solution was enhanced up to 2.8-fold in transgenic tobacco lines and by up to about 40% in two microalgae accessions. The present work provides new evidence for metabolic improvement of green organisms through the transgenic approach to remediation.     

表达多肽抗生素apidaecin的转基因烟草及其抗病性的初步分析

将多肽抗生素apidaecin基因与病程相关蛋白的信号肽序列融合,构建了apidaecin的分泌型植物表达载体、apidaecin与另一多肽抗生素Shiva\|I的双价分泌型植物表达载体,以本实验室原来构建的Shiva-I分泌型植物表达载体做对照,转化了模式植物烟草。对3种转基因植物进行了分子检测,转化再生苗95%为PCR阳性,Southern杂交结果进一步证明外源基因已经整合到了烟草基因组中,RT-PCR检测表明外源基因可以在转基因烟草内正常转录。对T0代转基因烟草进行烟草野火病的抗病性实验,从3种转基因烟草中都得到了抗病植株,病情指数分析的初步结果显示,双价转基因烟草抗病性最好,apidaecin的次之,Shiva-I的最差。     

A high sensitivity amperometric biosensor using a monomolecular layer of laccase as biorecognition element

Laccases from various sources were tested, and laccase from Rigidoporus lignosus was found to be the most active towards syringaldazine and ABTS, which are typical substrates of this class of enzymes, and towards the phenols found in olive oil mill wastewaters. This laccase was covalently immobilised by carbodiimide chemistry, on a self-assembled monolayer of 3-mercaptopropionic acid deposited on a gold surface. A flow biosensor, using the monolayer of laccase as bioelement and a glassy carbon electrode as amperometric transduction system, was developed. Although the amount of the immobilised enzyme (about 140 ng/cm2 effective surface area) was tiny, the biosensor showed a sensitivity of 3 nA/microM when 1,4-hydroquinone was used as substrate, and a half-life of 35 days. The proposed device permits detection of phenols in aqueous solutions at concentrations in the low micromolar range, i.e. below European Community limits. The biosensor was successfully used to detect phenols in wastewaters from an olive oil mill after minimal sample preparation (incubation of the aqueous sample with sodium borohydride for a few minutes) to suppress the current due to oxidised compounds present in the wastewaters.     

Expression of insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers the cold tolerance to transgenic tobacco

To elucidate the function of antifreeze protein from Microdera puntipennis dzhungarica for freezing stress tolerance in plant, the construct of MpAFP149 gene with the signal peptide sequence responsible for secreting the native MpAFP149 into the apoplast space under control of a cauliflower mosaic virus 35S promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. The observation of immunogold localization by TEM (transmission electron microscope) showed that the heterologous MpAFP149 protein was mainly distributed on the cell wall in apoplast of the transgenic tobacco plant. T1 generation transgenic tobacco plants displayed a more frost resistant phenotype and kept the lower ion leakage ratio and MDA (malondialdehyde) content in the leaves compared with wild-type ones at -1 degrees C for 3 days. The results showed that MpAFP149 provided protection and conferred cold tolerance to transgenic tobacco plant during freezing stress.     

Antimicrobial activity of olive mill wastewaters (alpechin) and biotransformed olive oil mill wastewater

Olive oil extraction produces a great volume of residue. These olive mill wastes are known as alpechin. This wastewater is a powerful pollutant, resistant to degradation and presents a severe environmental problem related to its high organic content made up largely of simple phenolic compounds, that have been described as being both antimicrobial and phytotoxic. This paper reviews briefly the antimicrobial activity of olive mill wastewaters and provides evidence to show the potential of micro organisms (Bacillus pumilus) to reduce the phenol content of alpechin, and also that biotransformation depends on the dilution (v/v) of the alpechin. Furthermore, we sought to provide a real evaluation of the extent of alpechin biotransformation. This was achieved by means of an internal reference, i.e. in relative terms, the phenol content resulting from the biotransformation process. The phenol content was measured using HPLC techniques, and results were obtained showing that the bacterium had most effect in reducing the phenol content of alpechin at concentrations of between 40 and 100%. It was also observed that at concentrations of 80%, in addition to a slight reduction in total phenols, new phenolic compounds, not present in the original alpechin, were generated.     

Expression of giant silkmoth cecropin B genes in tobacco

Cecropin B is a small antibacterial peptide from the giant silkmothHyalophora cecropia. To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion. All constructs were cloned in a plant expression vector and introduced in tobacco viaAgrobacterium tumefaciens. A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels. Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide. In none of the transgenic plants could the cecropin B peptide be detected. This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts. This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation. Nevertheless, transgenic tobacco plants were evaluated for resistance toPseudomonas solanacearum, the causal agent of bacterial wilt of many crops, andP. syringae pv.tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin Bin vitro. No resistance was found. These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.     

Antisense-overexpression of the MsCOMT gene induces changes in lignin and total phenol contents in transgenic tobacco plants

Initially, we isolated the caffeic acid O-methyltransferase (COMT) gene from Miscanthus sinensis (accession number HM062766.1). Next, we produced transgenic tobacco plants with down-regulated COMT gene expression to study its control of total phenol and lignin content and to perform morphological analysis. These transgenic plants were found to have reduced PAL and ascorbate peroxidases expression, which are related to the phenylpropanoid pathway and antioxidant activity. The MsCOMT-down-regulated plants had decreased total lignin in the leaves and stem compared with control plants. Reduced flavonol concentrations were confirmed in MsCOMT-down-regulated transgenic plants. We also observed a morphological difference, with reduced plant cell number in transgenic plants harboring antisense MsCOMT. The transgenic tobacco plants with down-regulated COMT gene expression demonstrate that COMT plays a crucial role related to controlling lignin and phenol content in plants. Also, COMT activity may be related to flavonoid production in the plant lignin pathway.     

Signal peptide of potato PinII enhances the expression of Cry1Ac in transgenic tobacco

The modified Cry l Ac was expressed in transgenic tobacco plants. To allow secretion of the CrylAc protein into the intercellular space, the signal peptide sequence of potato proteinase inhibitor II (pinII) was N-terminally fused to the CrylAc encoding region. Expression of Cry 1 Ac in transgenic tobacco plants was assayed with ELISA. The results showed that pinII signal peptide sequence enhanced the expression of Cry lAc protein and led to the secretion of the Cry 1 Ac protein in transgenic tobacco plants. GFP gene was also fused to the signal peptide sequence and transformed to tobacco. The results of fluorescent detection showed that GFP had localized in the apoplast of transgenic plants.     

Signal Peptide of Potato PinII Enhances the Expression of Cry1Ac in Transgenic Tobacco

Insect-resistant plants have been developed throughexpression of insecticidal proteins from Bacillusthuringiensis (Bt) in the early 1980s [1,2]. However, forcontrol of insect pests, it is necessary to increase theexpression of Bt protein overall or in specific plant tissues.To increase the expression level, synthetic Bt genes havebeen developed and used to produce transgenic plants[2–5]. A number of approaches have been taken to increasethe expression level of foreign proteins in transgeni…     

通过细胞内的靶向定位大幅度提高外源蛋白在转基因植株的积累水平

通过体外操作,对豇豆胰蛋白酶抑制剂(cpti)基因进行修饰,获得了一个融合蛋白基因(sck).该基因是在cpti基因的基础上,在其5'端添加了信号肽编码序列,在3'端添加了内质网滞留信号编码序列,旨在引导基因转译产物进入细胞内质网,并最终滞留在内质网及其衍生的蛋白体内.用sck基因转化烟草(Nicotiana tabacum L.),对获得的转基因植株进行ELISA检测.结果表明,含有修饰基因的转基因烟草CpTI蛋白含量有明显提高,比转未修饰cpti基因烟草平均高出2倍,最高单株可达4倍以上,同时转基因植株的抗虫性也有了显著的提高.结果表明,采用外源蛋白靶向定位的策略,可大幅度提高外源蛋白在转基因植物细胞内的积累量,在植物基因工程研究中具有广泛的借鉴意义.     

Detoxification of olive mill wastewaters by Moroccan yeast isolates

A total of 105 yeast strains were isolated from Moroccan olive oil production plants and evaluated for their ability to grow in olive oil mill wastewaters (OMW). The 9 isolates that grew best on OMW were selected for further study to evaluate their effect on removal of organic pollutants and OMW phytotoxicity (barley seed germination test). The results showed that at least four yeast isolates effectively lowered the toxicity of this effluent in addition to providing very useful materials in terms of both yeast biomass (6 g/l DW) and an irrigation fluid. This group of yeast isolates significantly reduced the concentration of total phenols (44% removal) and Chemical Oxygen Demand, COD (63% removal). The best germination rate of 80% for undiluted OMW was obtained for strain Candida holstii that also increased the pH from 4.76 to 6.75. Principal component analysis of the results obtained for the best yeast strains confirmed the importance of COD and total phenol reduction along with increase of organic nitrogen and final pH for the improvement of germination rates and phytotoxic reduction. This study has highlighted the potential of indigenous yeasts in detoxification of olive mill wastewaters.     

小黑杨PsnHB22基因在烟草中的遗传转化研究

HD-Zip转录因子在光信号转导、非生物胁迫、叶片发育等方面发挥重要的作用，HB22转录因子是HD-ZipⅠ亚家族的成员之一。为研究PsnHB22基因的功能，从小黑杨（Populus simonii×P.nigra）cDNA中克隆PsnHB22基因并构建植物表达载体进行烟草（Nicotiana tabacum）的遗传转化，以获得该基因过量表达的转基因株系。对转基因株系进行PCR、qRT-PCR分子检测后观察表型，结果显示在营养生长时期，转基因烟草叶片窄小并且株高显著低于野生型对照。测定转基因烟草及野生型叶片的叶绿素含量，发现转基因烟草叶绿素含量显著高于野生型。由此推测PsnHB22基因在植株高生长、光合作用及叶片的形态建成等过程中起着重要的作用。     

Transgenic tobacco expressing fungal laccase promotes the detoxification of environmental pollutants

The phytoremediation of soils contaminated with organic pollutants offers a low-cost method for removal of such pollutants. We have attempted to enhance the environmental decontamination functions of plants by introducing appropriate enzymatic activities from microorganisms. In the present study, we introduced an extracellular fungal enzyme, the laccase of Coriolus versicolor, into tobacco plants. One transgenic plant, designated FL4, produced laccase that was secreted into the rhizosphere. FL4 was able to remove 20 mol bisphenol A or pentachlorophenol per gram dry weight. The efficiency of this removal was apparently greater than that of control lines. Our results should stimulate efforts to develop plant-based technologies for the removal of environmental pollutants from contaminated environments.     

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.     

Biodegradation of olive oil mill wastewaterby Coriolus versicolor and Funalia trogii:effects of agitation,initial COD concentration,inoculum size and immobilization

The biodegradation of olive oil mill wastewater (OOMW) by Coriolus versicolor and Funalia trogii was investigated. Initial COD concentration, agitation and inoculum size were all found to be significant for biodegradation. Adding glucose, sulphate or nitrogen had no effect on biodegradation. During growth in optimum conditions, C.versicolor removed approximately 63% COD, 90% phenol and 65% colour within 6 days and F. trogii removed approximately 70% COD, 93% phenol and 81% colour of the OOMW used. The fungi also excreted large amounts of extracellular laccase into the medium. High biodegradation yields were also obtained by fungi immobilized in calcium alginate gels.     

Biodegradation of olive oil mill wastewaterby Coriolus versicolor and Funalia trogii:effects of agitation, initial COD concentration, inoculum size and immobilization

The biodegradation of olive oil mill wastewater (OOMW) by Coriolus versicolor and Funalia trogii was investigated. Initial COD concentration, agitation and inoculum size were all found to be significant for biodegradation. Adding glucose, sulphate or nitrogen had no effect on biodegradation. During growth in optimum conditions, C.versicolor removed approximately 63% COD, 90% phenol and 65% colour within 6 days and F. trogii removed approximately 70% COD, 93% phenol and 81% colour of the OOMW used. The fungi also excreted large amounts of extracellular laccase into the medium. High biodegradation yields were also obtained by fungi immobilized in calcium alginate gels.     

通过细胞内的靶向定位大幅度提高外源蛋白在转基因植株的积累水平

通过体外操作,对豇豆胰蛋白酶抑制剂(cpti)基因进行修饰,获得了一个融合蛋白基因(sck)。该基因是在cpti基因的基础上,在其5’端添加了信号肽编码序列,在3’端添加了内质网滞留信号编码序列,旨在引导基因转译产物进入细胞内质网,并最终滞留在内质网及其衍生的蛋白体内。用sck基因转化烟草(Nicotiana tabacum L.),对获得的转基因植株进行ELISA检测。结果表明,含有修饰基因的转基因烟草CpTI蛋白含量有明显提高,比转末修饰cpti基因烟草平均高出2倍,最高单株可达4倍以上,同时转基因植株的抗虫性也有了显著的提高。结果表明,采用外源蛋白靶向定位的策略,可大幅度提高外源蛋白在转基因植物细胞内的积累量,在植物基因工程研究中具有广泛的借鉴意义。