I. V. Goldenkova

[A thermostable Clostridium thermocellum lichenase-based reporter system for studying the gene expression regulation in prokaryotic and eukaryotic cells].

A new reporter system was developed to study the gene expression regulation in prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells. The system was based on the modified bacterial lichenase gene (licBM2), which was shown to meet the requirements for a reporter. The gene product was active and did not undergo modification in heterologous hosts. Simple and sensitive methods were used to detect and to quantitate the lichenase activity. Inducible licBM2 expression was demonstrated with E. coli and yeast cells, allowing the system to be employed in dynamic studies.

Preparation and properties of Clostridium thermocellum lichenase deletion variants and their use for construction of bifunctional hybrid proteins.

Major properties (pH and temperature optimum, stability) of lichenase (b-1,3-1,4-glucanase) deletion variants from Clostridium thermocellum were comparatively studied. The deletion variant LicBM2 was used to create hybrid bifunctional proteins by fusion with sequences of the green fluorescent protein (GFP) from Aequorea victoria. The data show that in hybrid proteins both GFP and lichenase retain their major properties, namely, GFP remains a fluorescent protein and the lichenase retains activity and high thermostability. Based on the results of this investigation and results that have been obtained earlier, the use of the deletion variants of lichenase and the bifunctional hybrid proteins as reporter proteins is suggested.

Expression of a Thermostable Bacterial Cellulase in Transgenic Tobacco Plants

The bacterial gene of the thermostable endo-β-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.

Expression of hybrid cry3aM–licBM2 genes in transgenic potatoes (Solanum tuberosum)

The full-modified Bacillus thuringiensis cry3a (cry3aM) gene was designed and synthesized for effective expression in plants. A plant expression vector pC29RBCS-leader-cry3aM–licBM2 was constructed for potato transformation. In this vector, the cry3aM sequence was fused in reading frame with a new reporter gene (licBM2) and a leader sequence for the rbcs gene. The reporter gene encoded thermostable lichenase and the leader sequence encoded a signal peptide for transporting protein product to chloroplasts. The vector contained the light-inducible promoter for rbcs gene isolated from Arabidopsis thaliana. Transgenic plants were obtained by Agrobacterium mediated transformation using microtuber explants. Transgenic plantlets were selected by kanamycin resistance and confirmed as transgenic by PCR with specific primers, evaluation of lichenase activity, and bioassay of Colorado potato beetle neonate larvae. Promoter activity assays under light induction (kinetic analysis) using lichenase activity and bioassay both showed high and stable expression of hybrid genes in transgenic plantlets. Furthermore, the presence of lichenase as a reporter protein in the composition of hybrid protein was shown to facilitate selection and analysis of the expression level of hybrid genes in transgenic plants.

[Bifunctional reporter genes: construction and expression in prokaryotic and eukaryotic cells].

Bifunctional reporter proteins were constructed to combine Clostridium thermocellum lichenase (LicBM2) with Aequorea victoria green fluorescent protein (GFP) or with Escherichia coli β-glucuronidase (GUS). The major properties of the initial proteins were preserved in the hybrid ones: LicBM2 was active at 65°C, GFP fluoresced, and GUS hydrolyzed its substrates. LicBM2 remained active after extension of its C or N end. Bifunctional reporter systems were shown to provide a convenient tool for studying the gene expression regulation in prokaryotic (E. coli) and eukaryotic (Saccharomyces cerevisiae, mammalian) cells, advantages of one reporter compensating for drawbacks of the other.

Modification of the Sunflower Defensin SD2 Gene Sequence and Its Expression in Bacterial and Yeast Cells

To achieve broader range of the defensin antimicrobial activity, based on the sd2 gene sequence, the modified gene, sd2mod, was constructed. Hybrid genes, sd2-licBM2, licBM2-sd2, licBM2-sd2mod, and sd2mod-licBM2, in which the wild-type and modified gene sequences were fused in frame with the reporter gene encoding thermostable lichenase, were constructed. Expression of the wild-type, modified, and hybrid genes was examined in the cells of pro- and eukaryotes. It was demonstrated that these genes were efficiently expressed in the cells of lower eukaryotes, the yeast. Inhibiting effect of the SD2 and SDmod proteins as the components of the hybrid proteins, SD2-LicBM2 and SD2mod-LicBM2, on the growth of the Fusarium culmorum hyphae was similar to that of the wild-type and modified proteins. It was shown that the presence of lichenase in the hybrid proteins facilitated selection and analysis of the hybrid proteins expression in transgenic organisms.

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

Expression of the bacterial gene for thermostable β-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 β-1,4-glucanases in plant physiological processes.

Exploring the Properties of Thermostable Clostridium thermocellum Cellulase CelE for the Purpose of Its Expression in Plants

The main properties (pH and temperature range, stability, substrate specificity) of the modified cellulase CelE (endo-β-1,4-glucanase) from Clostridium thermocellum have been analyzed with the goal of its expression in plants. The modified enzyme is similar to plant cellulases. Deletions in the N-terminus of the enzyme do not affect its biochemical properties. Based on the present investigation, we conclude that the modified β-1,4-glucanase CelEM1, when expressed in plants, will be a good model to study the role of cellulases in plants.

Physiological and biochemical characteristics of tobacco transgenic plants expressing bacterial dioxygenase

Expression of the 1,2-dihydroxynaphthalene dioxygenase (nahC) gene from Pseudomonas putida in tobacco transgenic plants produces notable phenotypic and biochemical changes: retarded growth and rooting and earlier flowering; chlorotic and necrotic spots on leaves; and a threefold increase in the total phenolics in the leaves of 6-week-old plants (94.51 μg/g fr wt as compared to 33.18 μg/g fr wt in the control) and in the phenylalanine-ammonia lyase activity in 4-week-old plants (0.035 U/g fr wt as compared to 0.014 U/g in the control plants of the same age). The transgenic plants expressing the nahC bacterial gene may serve as a model to study the putative functions of dioxygenases and phenol compounds in plant growth, development, and stress responses.

The Expression of the Bacterial Gene for Xylose(Glucose) Isomerase in Transgenic Tobacco Plants Affects Plant Morphology and Phytohormonal Balance

The gene encoding xylose(glucose) isomerase (P00944, EC 5.3.1.5) in Escherichia coli was put under the control of the 35S CaMV promoter and transferred to Nicotiana tabacum L. plants using an Agrobacterium tumefaciens vector. Transgenic plants, which synthesized an active bacterial enzyme, were characterized by the accelerated development of the root system, more rapid accumulation of total plant weight, and larger leaves. These changes were correlated with a changed hormonal balance and a changed activity of the chloroplast-gene expression.