Molecular cloning,characterization and expression of an elongation factor 1α gene in maize

A cDNA (zmEF1A) and the corresponding genomic clone (zmgEF1A) of a member of the gene family encoding the subunit of translation elongation factor 1 (EF-1) have been isolated from maize. The deduced amino acid sequence is 447 residues long interrupted by one intron. Southern blot analysis reveals that the cloned EF-1 gene is one member out of a family consisting of at least six genes. As shown by northern hybridizations in leaves the mRNA level increases at low temperature whereas time-course experiments over 24 h at 5°C show that in roots the overall mRNA level of EF-1 is transiently decreased. These results indicate that the expression of EF-1 is differently regulated in leaves and roots under cold stress.     

Molecular cloning and characterization of gene coding for γ-tocopherol methyltransferase from lettuce (Lactuca sativa)

γ-tocopherol methyltransferase is an important rate-limiting enzyme involved in tocopherol biosynthesis. The full-length cDNA encoding γ-tocopherol methyltransferase (designated as LsTMT) was cloned from Lactuca sativa for the first time by rapid amplification of cDNA ends and characterized by means of quantitative RT-PCR. The full-length cDNA of LsTMT was 1131 bp, with an open reading frame of 897 bp encoding a γ-tocopherol methyltransferase protein of 298 amino acids, with a calculated molecular mass of 33.06 kDa and an isoelectric point of 5.86. Comparative analysis revealed that LsTMT has a close similarity with γ-TMTs from other plant species. Bioinformatic analysis indicated that LsTMT shares a common evolutionary origin based on sequence similarity and has the closest relationship to γ-TMT from the sunflower, Helianthus annuus. Based on quantitative RT-PCR analysis, we found that expression of LsTMT is induced and strengthened by oxidative stresses such as strong light and drought. The cloning and characterization of LsTMT will be helpful to further understanding its role in the tocopherol biosynthesis pathway. We consider it to be a candidate gene for metabolic engineering of vitamin E in vegetable crops.     

Metabolic regulation of α-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.)

Expression of two genes in the -amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice -amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene.     

Effect of foliar spray of zinc on chloroplast β-carbonic anhydrase expression and enzyme activity in rice (Oryza sativa L.) leaves

The β-carbonic anhydrase (β-CA) is regarded as a zinc-containing enzyme involved in photosynthesis. Here, the rice plants of cv. N22 were treated with foliar spray at a Zn²⁺ concentration range from 0–35.0 mM during the tillering stage. The β-CA expression in the treated leaves was quantitatively determined by RT-qPCR and gel-based immunoblotting techniques, and its enzyme activity and relative chlorophyll concentration were measured. Results indicated that exogenous zinc could benefit rice plants at the tillering stage, particularly chloroplast β-CA with a fourfold enhancement in gene expression and a 14.6 % increase in its activity by treating the rice leaves with the 7.0-mM Zn²⁺ concentration, thereby promoting photosynthesis by a 19.4 % increase in relative chlorophyll concentration per unit leaf area. Results also showed that the application of Zn²⁺ at a concentration exceeding 7.0 mM could result in leaf senescence, and in some cases leaf hurts with significant inactivation (decreasing by approximately 70 %) of β-CA enzyme. It could be concluded that the application of 7.0-mM Zn²⁺ benefits rice plants at the tillering stage. The β-CA activity was associated with the catalytic microenvironment, thus providing an indicator for physiological response to exogenous zinc in rice.     

Molecular evolution and functional divergence of HAK potassium transporter gene family in rice （Oryza sativa L.）

The high-affinity K＋ （HAK） transporter gene family is the largest family in plant that functions as potassium transporter and is important for various aspects of plant life. In the present study, we identified 27 members of this family in rice genome. The phylogenetic tree divided the land plant HAK transporter proteins into 6 distinct groups. Although the main characteristic of this family was established before the origin of seed plants, they also showed some differences between the members of non-seed and seed plants. The HAK genes in rice were found to have expanded in lineage-specific manner after the split of monocots and dicots, and both segmental duplication events and tandem duplication events contributed to the expansion of this family. Functional divergence analysis for this family provided statistical evidence for shifted evolutionary rate after gene duplication. Further analysis indicated that both point mutant with positive selection and gene conversion events contributed to the evolution of this family in rice.     

Molecular cloning and characterization of RGA1 encoding a G protein α subunit from rice (Oryza sativa L. IR-36)

A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library prepared from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 M [adenylate-³²P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.     

Molecular cloning and differential expression patterns of the regulatory subunit B′ gene of PP2A in goldfish, Carassius auratus

It is well established that the protein serine/threonine phosphatase 2A (PP2A) plays very important roles in many different cellular processes, including cell proliferation and differentiation, gene expres-sion, neurotransmission, apoptosis, and aging. PP2A consists of three heterogenic subunits: the scaffold subunit A, the catalytic subunit C, and the regulatory subunit B. While both the scaffold and the catalytic subunits contain only two forms, at least four families of the regulatory subunits, B, B, B′′, and B′′′ have been identified. These regulatory subunits from different families are encoded by different genes and bear other functions besides directing the specificity of PP2A. To study the functions of the regulatory subunits of PP2A in lower vertebrates, we have cloned the full-length cDNA sequence of the gene encoding the regulatory subunit B′δ of PP2A from gold fish, Carassius auratus using 3′-RACE and 5′-RACE cloning strategies. Our results revealed that the full-length B′δ cDNA contains 2415 bp and encodes a protein of 555 amino acids. The B′δ protein displays a very high level of sequence identity with the B′δ regulatory subunit from other species of vertebrates. Regarding its expression pattern, RT-PCR revealed that the highest level of mRNA was detected in brain, a less level detected in liver, spermary, ovary, kidney and gill, and the lowest level detected in the fin. During different developmental stages of gold fish, the highest level of mRNA expression was detected at the stages of two-cell, mul-tiple-cell, blastula and gastrula, and a decreased level of B′δ ?mRNA was detected in other develop-mental stages. At the protein level, the highest expression level of B′δ protein was found in spermary, ovary, brain and heart, a less amount found in liver and the lowest level detected in kidney, gill and fin. Developmentally, B′δ protein was strongly expressed at the stages of two-cell, multiple-cell, blastula, gastrula, neurula, and optic vesicle, and then decreased at the stages of brain differentiation and eye pigmentation. These results suggest that B′δ appears to play a very important role during gold fish development and also in adult tissue homeostasis.     

Molecular cloning and expression of an expansin-like gene in ‘Navel’ orange fruit during postharvest stresses

In a search for differentially expressed genes in peel pitting of ‘Navel’ orange fruit (Citrus sinensis L. Osbeck), a cDNA subtraction library was constructed and a sequence encoding expansin-like gene was isolated and identified as pitting related gene. Based on sequence information derived from this fragment, a full-length cDNA (CsEXP, GenBank accession no. FJ769424) of 1,083 nucleotides encoding expansin was isolated from ‘Navel’ orange by RACE approaches. CsEXP encoded a protein of 254 amino acid residues with an open reading frame located in the region between 52 and 816 bp. The calculated molecular weight of the mature protein was 27.05 kDa and theoretical isoelectric point was 7.93. The deduced protein contained conserved domains of expansin: the histidine-phenylalanine-aspartate motif in central portion, cysteine residues in N-terminus, and tryptophan residues in C-terminal region. The expression of CsEXP was higher in pitting than the control. Exposure of fruit to stresses, including wounding, anoxia, low temperature (4°C), and treatment with ethylene, increased CsEXP mRNA levels in comparison with the control untreated fruit, whereas high temperature (40°C) decreased its mRNA levels. Since low temperature, low oxygen and wounding were suspected factors inducing peel pitting of citrus fruit. The present results provided us a clue that CsEXP may play a role in response to peel pitting related stresses.     

Molecular cloning and expression analysis of three genes encoding starch synthase II in rice

Three starch synthase (SS) genes, OsSSII-1, OsSSII-2 and OsSSII-3, were identified in rice (Oryza sativa L.) and localized to chromosomes 10, 2 and 6, respectively. The three OsSSII full-length cDNAs were cloned, and the predicted amino acid sequences were found to share 52–73% similarity with other members of the plant SSII family. The SS activity of each OsSSII was confirmed by expression and enzyme activity assay in Escherichia coli. Expression profile analysis revealed that OsSSII-1 was expressed in endosperms, leaves and roots; OsSSII-2 was mainly expressed in leaves, while OsSSII-3 was mainly expressed in endosperms. Similar to the OsSSI proteins, the OsSSII-2 and OsSSII-3 proteins were found in the soluble as well as the starch-granule-bound fractions in rice. The roles of the OsSSII proteins in starch biosynthesis in rice and the evolutionary relationships of the genes encoding monocotyledonous and dicotyledonous class-II SS enzymes are discussed.Abbreviations CDS Coding domain sequence - EST Expressed sequence tag - GB Granule-bound - Glc Glucose - SS Starch synthase     

Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

Molecular cloning and expression of 17β-hydroxysteroid dehydrogenase type 2 gene in Hu sheep

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyzes the NADP+-dependent oxidation of the most potent estrogen 17β-estradiol into the weak estrogen estrone, and the conversion of testosterone to androstenedione. It has been reported that 17β-HSD2 was expressed in many tissues in human, rats, however, the full-length sequence of 17β-HSD2 gene and its expression in ewe were still unknown. In this study, we cloned the full-length cDNA sequence and investigated mRNA differential expression in 28 tissues of 12 adult Hu-Sheep which were fed with high- and low- dietary intake. The 1,317 bp full-length cDNA sequence was first cloned. The coding region was 1,167 bp in length, and the monomer was estimated to contain 389 amino acid residues. It shares high AA sequence identity with that of bos Taurus (96.13 %), sus scrofa (77.06 %), canis lupus familiaris (70.44 %), Callithrix jacchus (65.72 %), Nomascus leucogenys (65.46 %), pan troglodytes (65.21 %), human (64.69 %), mus musculus (58.35 %), and a comparatively lower identity to danio rerio (37.85 %). 17β-HSD2 gene was high expressed in gastrointestinal (GI) tract, liver, but weakly expressed in other tissues. No detected expression was examined in lung. 17β-HSD2 gene expression was significantly difference in rumen, omasum, duodenum, cecum, hypophysis after high- and low- dietary intake. Results from the present study suggested that 17β-HSD2 plays a crucial role in almost all tissues protecting against excessive levels of active steroid hormone, and GI tract maybe an important steroid hormone metabolizing organ in Hu-Sheep. This present study is the first to provide the primary foundation for further insight into this ovine gene.     

Molecular cloning of the β-cyclodextrin synthetase gene from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis

Summary The gene for -CGTase from an alkalophilic bacterium, Bacillus sp. #1011, was cloned in an Escherichia coli phage D69 and recloned in an E. coli plasmid pBR322 and a B. subtilis plasmid pUB110. An E. coli recombinant plasmid pTUE202 and a B. subtilis plasmid pTUB703 were selected from ten plasmids, because the transformants by each of the two plasmids produced the highest amount of extracellular -CGTase in each strain. The plasmids were stably maintained and expressed in each bacterial strain. A common DNA region of approximately 2.5 kb was defined in the ten plasmids, and the enzymatic activity was lost when a part of the common region was deleted. The major product of hydrolysis from starch by the -CGTases of E. coli [pTUB202] and B. subtilis [pTUB703] was -CD as in the case of the enzyme of the parental Bacillus sp. #1011.Abbreviations -CGTase -cyclodextrin synthetase - -CD -cyclodextrin - -CD -cyclodextrin - -CD -cyclodextrin - [] designates plasmid-carrier state     

Molecular cloning,expression and structure of the endo-1,5-α-l-arabinase gene of Aspergillus niger

Secretion of endo-1,5--l-arabinase A (ABNA) by an Aspergillus niger xylulose kinase mutant upon mycelium transfer to medium containing l-arabitol was immunochemically followed with time to monitor its induction profile. A cDNA expression library was made from polyA ⁺ RNA isolated from the induced mycelium. This library was immunochemically screened and one ABN A specific clone emerged. The corresponding abnA gene was isolated from an A. niger genomic library. Upon Southern blot analysis, a 3.1-kb HindIII fragment was identified and subcloned to result in plasmid pIM950. By means of co-tranformation using the A. niger pyrA gene as selection marker, the gene was introduced in both A. niger and A. nidulans uridine auxotrophic mutants. Prototrophic A. niger and A. nidulans transformants overproduced A. niger ABN A upon growth in medium containing sugar beet pulp as the sole carbon source, thereby establishing the identity and functionality of the cloned gene. The DNA sequence of the complete HindIII fragment was determined and the structure of the abnA gene as well as of its deduced gene product were analysed. Gene abnA contains three introns within its structural region and codes for a protein of 321 amino acids. Signal peptide processing results in a mature protein of 302 amino acids with a deduced molecular mass of 32.5 kDa. A. niger abnA is the first gene encoding an ABN to be isolated and characterized. Correspondence to: L. H. de Graaff     

Chromosomal localization and genomic organization of α-amylase genes in rice (Oryza sativa L.)

Summary Genes for -amylase, alcohol dehydrogenase, andEm, an ABA-regulated gene expressed late in embryogenesis, were localized on rice chromosomes by the analysis of primary trisomies. The validity of the mapping approach was confirmed usingAdh-1 as a control. TheAdh-1 gene has previously been assigned to chromosome 11 using conventional techniques. In this study we confirm this assignment and report an additional locus for alcohol dehydrogenase (Adh-2) on chromosome 9. The -amylase genes were located on chromosomes 1, 2, 6, 8, and 9 while theEm gene was mapped to chromosome 5. To facilitate trisomic analysis and correlation of cloned genes with bands observed on Southern blots, a nomenclature for the rice -amylase genes has been proposed. In addition to mapping nine cloned -amylase genes, we have identified two previously uncloned -amylase genes as part of this study. Polymorphism for -amylase genes belonging to each of the three subfamilies was observed between M202 and IR36. The maximum degree of polymorphism was found among genes belonging to the RAmy3 subfamily, which also has the most diverse group of genes.     

Molecular cloning,expression and characterization of Pekin duck interferon-λ

Interferons (IFNs) are the first line of defense against viral infections in vertebrates. Type III interferon (IFN-λ) is recognized for its key role in innate immunity of tissues of epithelial origin. Here we describe the identification of the Pekin duck IFN-λ ortholog (duIFN-λ). The predicted duIFN-λ protein has an amino acid identity of 63%, 38%, 37% and 33% with chicken IFN-λ and human IFN-λ3, IFN-λ2 and IFN-λ1, respectively. The duck genome contains a single IFN-λ gene that is comprised of five exons and four introns. Recombinant duIFN-λ up-regulated OASL and Mx-1 mRNA in primary duck hepatocytes. Our observations suggest evolutionary conservation of genomic organization and structural features implicated in receptor binding and antiviral activity. The identification and expression of duIFN-λ will facilitate further study of the role of type III IFN in antiviral defense and inflammatory responses of the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.