Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance

 Transformation of barley and wheat via particle bombardment with a gene derived from Vitis vinifera L. (Vst1 gene) resulted in the expression of the foreign phytoalexin, resveratrol, in the transformed plants. Transgenic barley plants were regenerated from microspores and transgenic wheat plants from immature embryos were both selected on Basta. Stable integration of the gene in the genomes of transgenic barley and wheat plants, as well as their progeny, was analysed by Southern-blot analysis. The induction of the stilbene synthase promoter and the transient expression of stilbene synthase-specific mRNA after induction by wounding and infection were proofed in T₁ and T₂ progeny plants. An enhanced expression of the Vst1 gene under control of the stilbene synthase promoter was observed with enhancer sequences from the cauliflower mosaic virus 35s (CaMV 35s) promoter. The enzyme activity of the stilbene synthase was analysed in T₁ progeny plants. The first pathological results indicated an increased resistance of transgenic barley plants to Botrytis cinerea used as a model experimental system. Received: 5 November 1997 / Accepted: 11 November 1997     

The use of a thermostable β-glucanase gene from Clostridium thermocellum as a reporter gene in plants

In order to take advantage of the high thermostability of its product, β-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine T_R-DNA 2′ gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering. Received: 4 March 1997 / Accepted: 8 October 1997     

Biofilm formation by haloarchaea

A fluorescence‐based live‐cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P‐value < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet‐like, multi‐layered biofilms containing micro‐ and macrocolonies (up to 50 μm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t‐ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757^T and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754^T and Hfx. volcanii DSM 3757^T, cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754^T showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.     

玉米胚胎发生后期丰富蛋白基因的启动子克隆与功能验证

该研究在生物信息学分析的基础上,克隆玉米胚胎发生后期丰富蛋白基因(MGL3)的启动子序列(pMGL3),进行非生物逆境应答元件分析以及实时定量PCR验证其非生物逆境胁迫响应特性,构建了pMGL3启动子驱动报告基因(GUS)表达载体,基因枪法转化玉米愈伤组织,通过GUS染色验证pMGL3启动子在非生物逆境胁迫下的驱动活性。再根据启动子序列分析结果,去除不同的顺式作用元件,构建不同长度pMGL3启动子驱动报告基因GUS表达载体,农杆菌介导法转化烟草叶盘,以确定pMGL3启动子的最短活性序列。结果显示:pMGL3启动子长1 554bp,存在多种与非生物逆境胁迫应答相关的调控元件,在干旱、高盐、低温胁迫及脱落酸、乙烯诱导下驱动MGL3基因增量表达,用以驱动GUS基因转化玉米愈伤组织,在高渗、高盐、低温胁迫及脱落酸诱导下具有驱动活性,且截短至325bp仍可保持驱动活性。研究表明,pMGL3启动子的确有非生物逆境诱导启动活性,进一步验证其作用机理后可运用于玉米抗逆转基因研究。     

Synthesis and assembly of a cholera toxin B subunit-rotavirus VP7 fusion protein in transgenic potato

A gene encoding VP7, the outer capsid protein of simian rotavirus SA11, was fused to the carboxyl terminus of the cholera toxin B subunit gene. A plant expression vector containing the fusion gene under control of the mannopine synthase P₂ promoter was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB::VP7 fusion gene was detected in the genomic DNA of transformed potato leaf cells by polymerase chain reaction (PCR) amplification methods. Immunoblot analysis of transformed potato tuber tissue extracts showed that synthesis and assembly of the CTB::VP7 fusion protein into oligomers of pentameric size occurred in the transformed plant cells. The binding of CTB::VP7 fusion protein pentamers to sialo-sugar containing GM1 ganglioside receptors on the intestinal epithelial cell membrane was quantified by enzyme-linked immunosorbent assay (ELISA). The ELISA results showed that the CTB::VP7 fusion protein made up approx 0.01% of the total soluble tuber protein. Synthesis and assembly of CTB::VP7 monomers into biologically active pentamers in transformed potato tubers demonstrates the feasibility of using edible plants as a mucosal vaccine for the production and delivery system for rotavirus capsid protein antigens.     

Monitoring dynamic expression of nuclear genes in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> by using a synthetic luciferase reporter gene

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii. This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii.     

Evaluation in tobacco of the organ specificity and strength of therolD promoter,domain A of the 35S promoter and the 35S2 promoter

In order to study the expression in plants of therolD promoter ofAgrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of thegusA (uidA) marker gene under control of tworolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R₁ progeny plants. In mature transformed tobacco plants, therolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity inrolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter,domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of therolD-gus genes was less than that of agus gene with a 35S promoter with doubled domain B, 35S²-gus. TherolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.     

Over-expression of a <Emphasis Type="Italic">LEA</Emphasis> gene in rice improves drought resistance under the field conditions

Late embryogenesis abundant (LEA) proteins have been implicated in many stress responses of plants. In this report, a LEA protein gene OsLEA3-1 was identified and over-expressed in rice to test the drought resistance of transgenic lines under the field conditions. OsLEA3-1 is induced by drought, salt and abscisic acid (ABA), but not by cold stress. The promoter of OsLEA3-1 isolated from the upland rice IRAT109 exhibits strong activity under drought- and salt-stress conditions. Three expression constructs consisting of the full-length cDNA driven by the drought-inducible promoter of OsLEA3-1 (OsLEA3-H), the CaMV 35S promoter (OsLEA3-S), and the rice Actin1 promoter (OsLEA3-A) were transformed into the drought-sensitive japonica rice Zhonghua 11. Drought resistance pre-screening of T₁ families at anthesis stage revealed that the over-expressing families with OsLEA3-S and OsLEA3-H constructs had significantly higher relative yield (yield under drought stress treatment/yield under normal growth conditions) than the wild type under drought stress conditions, although a yield penalty existed in T₁ families under normal growth conditions. Nine homozygous families, exhibiting over-expression of a single-copy of the transgene and relatively low yield penalty in the T₁ generation, were tested in the field for drought resistance in the T₂ and T₃ generations and in the PVC pipes for drought tolerance in the T₂ generation. Except for two families (transformed with OsLEA3-A), all the other families (transformed with OsLEA3-S and OsLEA3-H constructs) had higher grain yield than the wild type under drought stress in both the field and the PVC pipes conditions. No significant yield penalty was detected for these T₂ and T₃ families. These results indicate that transgenic rice with significantly enhanced drought resistance and without yield penalty can be generated by over-expressing OsLEA3-1 gene with appropriate promoters and following a bipartite (stress and non-stress) in-field screening protocol.     

Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes

An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a gene for -glucuronidase (GUS) with an intron fused to the CaMV 35S promoter. Inoculated calli were plated on medium that contained cefotaxime to eliminate bacteria. Four weeks later, transformed cells were selected on medium that contained 20 mg L^–1 hygromycin. A histochemical assay for GUS activity revealed that selection by hygromycin was complete after eight weeks. The integration of the T-DNA of the Ri-plasmid and pIG121Hm into the plant genome was confirmed by PCR. Plants derived from transformed calli were produced on half-strength MS medium supplemented with 0.1 mg L^–1 GA₃ after about 5 months of culture. The presence of the gusA, nptII, and rol genes in the genomic DNA of regenerated plants was detected by PCR and Southern hybridization, and the expression of these transgenes was verified by RT-PCR.     

Enhancement of the tolerance of Arabidopsis to high temperatures by genetic engineering of the synthesis of glycinebetaine

Arabidopsis thaliana was transformed with the codA gene for choline oxidase from Arthrobacter globiformis under control of the 35S RNA promoter of cauliflower mosaic virus. As a result, high levels of glycinebetaine accumulated in the seeds of transformed plants. Transformation with the codA gene significantly enhanced the tolerance to high temperatures during the imbibition and germination of seeds, as well as during growth of young seedlings. The extent of enhancement of the tolerance to high temperature was correlated with levels of choline oxidase expressed and of glycinebetine accumulated in the transformed plants. The induction of homologues of heat shock protein 70 at high temperature was less conspicuous in the transformed plants than in the wild-type plants, suggesting that the transformation alleviated the high-temperature stress.     

Expression of full-length bioactive antimicrobial human lactoferrin in potato plants

A cDNA fragment encoding human lactoferrin (hLF) linked to a plant microsomal retention signal peptide (SEKDEL) was stably integrated into the Solanum tuberosum genome by Agrobacterium tumefaciens-mediated leaf disk transformation methods. The lactoferrin gene was expressed under control of both the auxin-inducible manopine synthase (mas) P2 promoter and the cauliflower mosaic virus (CaMV) 35S tandem promoter. The presence of the hLF cDNA in the genome of regenerated transformed potato plants was detected by polymerase chain reaction amplification methods. Full-length hLF protein was identified by immunoblot analysis in tuber tissue extracts from the transformed plants by immunoblot analysis. The hLF produced in transgenic plant tissues migrated during polyacrylamide gel electrophoresis as a single band with an approximate molecular mass equal to hLF. Auxin activation of the mas P2 promoter increased lactoferrin expression levels in transformed tuber and leaf tissues to approximately 0.1% of total soluble plant protein. Antimicrobial activity against four different human pathogenic bacterial strains was detected in extracts of lactoferrin-containing potato tuber tissues. This is the first report of synthesis of full length, biologically active hLF in edible plants.     

Combined expression of chitinase and β-1,3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani

Agrobacterium-mediated transformation of rice was done using the binary vector pNSP3, harbouring the rice chitinase (chi11) gene under maize ubiquitin promoter and the tobacco β-1,3-glucanase gene under CaMV 35S promoter in the same T-DNA. Four of the six T₀ plants had single copies of complete T-DNAs, while the other two had complex integration patterns. Three of the four single-copy lines showed a 3:1 segregation ratio in the T₁ generation. Northern and western blot analyses of T₁ plants revealed constitutive expression of chitinase and β-1,3-glucanase genes. Homozygous T₂ plants of the single-copy lines CG20, CG27 and CG53 showed 62-, 9.6- and 11-fold higher chitinase activity over the control plants. β-1,3-Glucanase activity was 1.1- to 2.5-fold higher in the transgenic plants. Bioassay of homozygous T₂ plants of the three single-copy transgenic lines against Rhizoctonia solani revealed a 60% reduction in sheath blight Disease Index in the first week. The Disease Index increased from 61.8 in the first week to 90.6 in the third week in control plants, while it remained low (26.8–34.2) in the transgenic T₃ plants in the corresponding period, reflecting the persistence of sheath blight resistance for a longer period.     

Nitric oxide reduction, the last step in denitrification by Fusarium oxysporum, is obligatorily mediated by cytochrome P450nor

The involvement of cytochrome P450nor (P450nor) is the most striking feature of the fungal denitrifying system, and has never been shown in bacterial systems. To establish the physiological significance of the P450nor, we constructed and investigated mutants of Fusarium oxysporum that lacked the gene for P450nor. We mutated the gene by targeted integration of a disrupted gene into the chromosome of F. oxysporum. The mutants were shown to contain neither P450nor protein nor nitric oxide (NO) reductase (Nor) activity, implying that they are indeed deficient in P450nor. These mutants had apparently lost the denitrifying activity and failed to evolve nitrous oxide (N₂O) upon incubation under oxygen-limiting conditions in the presence of nitrate. Their mycelia exhibited normal levels of dissimilatory nitrite reductase (Nir) activity and were able to evolve NO under these conditions. The promoter region of the P450nor gene was fused to lacZ and introduced into the wild-type strain of F. oxysporum. The transformed strain produced β-galactosidase under denitrifying conditions as efficiently as the wild type does P450nor. These results represent unequivocal genetic evidence that P450nor is essential for the reduction of NO to N₂O, the last step in denitrification by F. oxysporum. Received: 28 June 1999 / Accepted: 22 December 1999     

Improvement of ligninolytic properties by recombinant expression of glyoxal oxidase gene in hyper lignin-degrading fungus Phanerochaete sordida YK-624

Glyoxal oxidase (GLOX) is a source of the extracellular H₂O₂ required for the oxidation reactions catalyzed by the ligninolytic peroxidases. In the present study, the GLOX-encoding gene (glx) of Phanerochaete chrysosporium was cloned, and bee2 promoter of P. sordida YK-624 was used to drive the expression of glx. The expression plasmid was transformed into a P. sordida YK-624 uracil auxotrophic mutant (strain UV-64), and 16 clones were obtained as GLOX-introducing transformants. These transformants showed higher GLOX activities than wild-type P. sordida YK-624 and control transformants harboring marker plasmid. RT-PCR analysis indicated that the increased GLOX activity was associated with elevated recombinant glx expression. Moreover, these transformants showed higher ligninolytic activity than control transformants. These results suggest that the ligninolytic properties of white-rot fungi can be improved by recombinant expression of glx.     

Expression of a humanS-adenosylmethionine decarboxylase cDNA in transgenic tobacco and its effects on polyamine biosynthesis

S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key regulatory enzyme in the polyamine biosynthetic pathway. Numerous studies have shown that the enzyme activity and polyamine levels are generally correlated with cellular growth in plants, animals and bacteria. In order to gain more insight into the role of polyamines in plants, human SAMDC cDNA under control of the 35S promoter of cauliflower mosaic virus, along with a neomycin phosphotransferase gene, was transferred to tobacco (Nicotiana tabacum cv. Xanthi) viaAgrobacterium tumefaciens. Transgenic plants showed the presence of human SAMDC mRNA and a 2-4-fold increase in SAMDC activity. In the transformed tissues, putrescine levels were significantly reduced, while spermidine content was 2–3 times higher than the control tissues. Cellular spermine content was either increased or remained unchanged. Excised leaf segments from transformed plants frequently produced shoots even on callus inducing medium.     

Transgenic Expression of Trypsin Inhibitor CMe from Barley in Indica and Japonica Rice,Confers Resistance to the Rice Weevil Sitophilus Oryzae

Indica and japonica rice (Oryza sativa L.) plants were transformed by particle bombardment with the Itr1 gene encoding the barley trypsin inhibitor BTI-CMe, under the control of its own promoter that confers endosperm specificity, and the maize ubiquitin promoter. From 38 independent transgenic lines of indica (breeding line IR58) and 15 of the japonica (cv Senia) selected, 22 and 11, respectively, expressed the barley inhibitor at detectable levels. The transgene was correctly translated as indicated by western blot analysis with a level of expression in R₃ seeds up to 0.31% (IR58) and 0.43% (Senia) of the total extracted protein. The functional integrity of BTI-CMe was confirmed by trypsin activity assays in liquid media and by activity staining gels, performed with seed extracts. The significant reduction of the survival rate of the rice weevil (Sitophilus oryzae, Coleoptera: Curculionidae) reared on homozygous transgenic indica and japonica rice seeds expressing the BTI-CMe, compared to non-transformed controls, and the decrease in the trypsin-like activity of insect crude midgut extracts, confirmed the utility of this proteinase inhibitor gene for the control of important storage pests.     

Chilling Tolerance of Potato Plants Transformed with a Yeast-Derived Invertase Gene under the Control of the B33 Patatin Promoter

Tolerance to chilling was compared under in vitro conditions in potato plants (Solanum tuberosum L., cv. Désirée) transformed with a yeast-derived invertase gene under the control of the B33 class 1 tuber-specific promoter (the B33-inv plants) and potato plants transformed only with a reporter gene (the control plants). The expression of the inserted yeast invertase gene was proved by following the acid and alkaline invertase activities and sugar contents in the leaves under the optimum temperature (22°C). The total activities of acid and alkaline invertases in the B33-inv plants exceeded those in the control plants by the factors of 2–3 and 1.3, respectively. In the B33-inv plants, the activity of acid invertase twice exceeded that of the alkaline invertase, whereas the difference equaled 12% in the control plants. The contents of sucrose and glucose increased in the B33-inv plants by 21 and 13%, respectively, as compared to the control. Chilling at +3 and –1°C for 1, 3, and 6 h did not affect the rate of lipid peroxidation, as measured by the content of malonic dialdehyde (MDA) in the leaves of the genotypes under study. Only the longer exposures (24 h at +3 and –1°C and 7 days at +5°C) produced a significant decline in the MDA content in the B33-invplants, as compared to the control. Following short freezing (20 min at –9°C), the content of MDA increased by 50% in the leaves of the control plants, while in the B33-inv plants, cold-treated and control plants did not differ in the MDA content. The authors presume that the potato plants transformed with the yeast invertase gene acquire a higher tolerance to low temperatures as compared to the control plants, apparently due to the changes in sugar ratio produced by the foreign invertase.