Cloning and characterization of a cDNA encoding Ran binding protein from wheat.

Ran, which functions in nucleocytoplasmic transport and mitosis, binds to and is regulated in part by Ran binding protein (RanBP). A RanBP cDNA (TaRanBP1) was isolated from a wheat cDNA library using RT-PCR product as a probe. The predicted amino acid sequence of TaRanBP1 is over 60% identity to AtRanBP1 from Arabidopsis and also with considerable similarity to human and fungi RanBPs. TaRanBP1 gene was expressed ubiquitously in roots, leaves and stems, with a similar abundance in these tissues. Phylogenetic reconstruction of TaRanBP1 with 32 other RanBPs from 26 species of organisms revealed that RanBPs from plants, animals and fungi clustered as the distinct groups, intraspecies isoforms were not developed for RanBPs, contrast with most other ancestral genes. Structural analysis revealed that all RanBPs were highly conserved in the middle region of their amino acid sequence, which included Ran binding domain and the three conserved motifs that have the essential roles in binding with Ran protein and promotion of GTP hydrolysis by the Ran/RanGAP/RanBP complex. However, N-terminus and C-terminus exhibited very low similarity between the different RanBPs. The different structures in N-terminus and C-terminus of RanBPs are likely to direct the Ran into the specific physiological processes and subsequently exhibit the different roles in different organisms.     

Cloning and characterization of a cDNA encoding bovine mannan-binding protein

To identify the bovine mannan-binding protein (MBP), a search for the cDNA homologue of human MBP was carried out. cDNA clones encoding bovine MBP were isolated from a bovine liver cDNA library using a cDNA fragment encoding a short collagen region, neck domain and carbohydrate recognition domain of human MBP. The cDNA carried an insert of 747 bp encoding a protein of 249 amino acid (aa) residues with a signal peptide of 19 aa. The mannan-binding protein fraction of bovine serum that eluted with 100 mM mannose from a mannan-Sepharose column was analyzed under reducing conditions by SDS-PAGE. The major band of 33 kDa obtained reacted with anti-human MBP rabbit serum. The partial aa sequence of the purified 33-kDa protein was identical to the aa sequence deduced from the obtained cDNA. Results of the passive hemolysis experiment using sheep erythrocytes coated with yeast mannan suggest that this MBP has the ability to activate complement. Northern blot analysis showed a 1.8-kb mRNA that was expressed only in the liver. Based on results of genomic analysis, this bovine MBP is likely to be a homologue of human MBP and to also have homology to rat and mouse MBP-C which are localized in liver cells rather than to rat and mouse MBP-A found in serum. Alignments of bovine collectins show that bovine MBP cannot be included among the other bovine collectins, such as bovine SP-D, conglutinin and CL-43. Finally, these genomic and biological analyses indicate that the cDNA obtained here encoded a bovine serum MBP.     

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coli. The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.     

Cloning and characterization of a cDNA encoding 14-3-3 protein with leaf and stem-specific expression from wheat.

The 14-3-3 proteins, originally described as the mammalian brain proteins, are ubiquitous eukaryotic proteins and have been shown to exert an array of function. A great number of 14-3-3 sequences have been reported in Eudicotyledon. The data of 14-3-3 from the monocotyledon plants, however, are limited. In this report, a 14-3-3 cDNA (designated as Ta14A) was isolated from wheat. An extensive search in GenBank database revealed another 14 14-3-3 isoforms from monocotyledonous plants. These proteins plus 14-3-3 isoforms from Arabidopsis were used for phylogenetic reconstruction, which revealed two groups of 14-3-3 proteins in monocotyledonous plants, namely epsilon and non-epsilon, respectively. The epsilon isoforms were present in monocotyledonous plants. Therefore, the gene duplication to result in an epsilon and non-epsilon isoforms was likely to take place before the speciation of monocotyledon and Eudicotyledon plants. Structural analysis indicated that the different conserved domains and structural characters existed in the monocotyledon 14-3-3 isoforms, which will affect their interaction with other effector proteins. Ta14A was strongly expressed in leaf and stem, undetected in root, suggesting it may have the unique functions within these tissues. These data suggest that structure difference and spatial expression of 14-3-3 will be the important factors to confine its functional specificity.     

Cloning and characterization of a cDNA encoding the wheat (Triticum durum Desf.) CM16 protein

A Triticum durum cDNA library prepared from developing endosperm (22 days after flowering (DAF)) was screened using synthetic oligonucleotide probes covering part of the CM3 and CM16 N-terminal protein sequences. A full-length cDNA clone (pTd78) encoding the CM16 protein (chloroform/methanol-soluble protein) was isolated and characterized. To our knowledge this is the first characterization of a clone coding for a wheat CM protein. The CM16 protein is synthesized as a preprotein with a signal peptide of 24 residues, the molecular weight of the mature protein being 13 438 Da. As other members of the cereal trypsin/-amylase inhibitor family, the CM16 protein contains 10 cysteine residues, their position being well conserved. In developing endosperm the highest level of CM16 mRNA was detected at mid-maturation.     

A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation

A novel cDNA clone, Tad1, was isolated from crown tissue of winter wheat after differential screening of cold acclimation-induced genes. The Tad1 cDNA encoded a 23kDa polypeptide with a potential N-terminal signal sequence. The putative mature sequence showed striking similarity to plant defensins or gamma-thionins, representing low molecular size antipathogenic polypeptides. High levels of Tad1 mRNA accumulation occurred within one day of cold acclimation in crown tissue and the level was maintained throughout 14 days of cold acclimation. Similar rapid induction was observed in young seedlings treated with low temperature but not with exogenous abscisic acid. In contrast to defensins from other plant species, neither salicylic acid nor methyl jasmonate induced expression of Tad1. The recombinant mature form of TAD1 polypeptide inhibited the growth of the phytopathogenic bacteria, Pseudomonas cichorii; however, no antifreeze activity was detected. Collectively, these data suggested that Tad1 is induced in cold-acclimated winter wheat independent of major defense signaling(s) and is involved in low temperature-induced resistance to pathogens during winter hardening.     

Cloning, expression and characterization of a cDNA encoding a lipase from Rhizopus delemar.

A lambda gt11 cDNA library was constructed in Escherichia coli using poly(A)-selected mRNA from the fungus, Rhizopus (Rp.) delemar. Lipase-producing members of the library were identified by means of a phenotypic score wherein the release of fatty acids by lipase causes a characteristic color change in the growth medium. One such isolate contained a 1287-bp insert (LIP cDNA) which hybridizes to 1.25- to 1.35-kb mRNA species from Rp. delemar. The lipase produced in E. coli containing the LIP cDNA exhibits the same substrate selectivity as the authentic fungal enzyme, hydrolyzing ester bonds at the stereospecific numbering (sn) sn-1 and sn-3, but not the sn-2, positions of triglycerides. The complete nucleotide sequence of the LIP cDNA was determined. By reference to the N-terminal sequence of authentic Rp. delemar lipase, the lipase-encoding region was identified within this fragment. The LIP cDNA encodes a putative preprolipase consisting of a 26-amino-acid(aa) signal sequence, a 97-aa propeptide, and a 269-aa mature enzyme. The predicted mature lipase has the same molecular weight and aa composition as that of Rp. delemar, is highly homologous to that produced by the fungus Rhizomucor miehei, and contains the consensus pentapeptide (Gly-Xaa-Ser-Yaa-Gly) which is conserved among lipolytic enzymes. It is concluded that the LIP cDNA is an essentially full-length analogue of the lipase-encoding gene of Rp. delemar. The lipase encoded by the LIP cDNA occupies a cytoplasmic location when synthesized in E. coli. Unprocessed forms of the lipase accumulate in E. coli.     

Isolation of a cDNA encoding a granule-bound 152-kilodalton starch-branching enzyme in wheat

Screening of a wheat (Triticum aestivum) cDNA library for starch-branching enzyme I (SBEI) genes combined with 5'-rapid amplification of cDNA ends resulted in isolation of a 4,563-bp composite cDNA, Sbe1c. Based on sequence alignment to characterized SBEI cDNA clones isolated from plants, the SBEIc predicted from the cDNA sequence was produced with a transit peptide directing the polypeptide into plastids. Furthermore, the predicted mature form of SBEIc was much larger (152 kD) than previously characterized plant SBEI (80-100 kD) and contained a partial duplication of SBEI sequences. The first SBEI domain showed high amino acid similarity to a 74-kD wheat SBEI-like protein that is inactive as a branching enzyme when expressed in Escherichia coli. The second SBEI domain on SBEIc was identical in sequence to a functional 87-kD SBEI produced in the wheat endosperm. Immunoblot analysis of proteins produced in developing wheat kernels demonstrated that the 152-kD SBEIc was, in contrast to the 87- to 88-kD SBEI, preferentially associated with the starch granules. Proteins similar in size and recognized by wheat SBEI antibodies were also present in Triticum monococcum, Triticum tauschii, and Triticum turgidum subsp. durum.     

Cloning,characterization and expression of a cDNA encoding a granulin-like polypeptide in Ciona savignyi

Previous study in our laboratory confirmed that a novel polypeptide, CS5931 derived from Ciona savignyi possesses potent antitumor activity. In the present study, the full length cDNA of CS5931 precursor, termed Cs-pgrn-1 was cloned. The complete cDNA sequence of this gene consists of 685 bp containing an open reading frame (ORF) of 522 bp (173 amino acid residues). In silico analysis revealed that the polypeptide consists of two identical domains, similar with granulin (GRN) found in other species, and each of the domain encodes a polypeptide identical with CS5931. Phylogenetic analysis confirmed that CS5931 shares high homology with Ciona intestinalis GRN and is conserved during evolution. The polypeptide also shows high similarity with human GRN A, B, and C. Prediction of 3D protein structure revealed the 3D structure of CS5931 is very similar with human GRN A. The CS5931 was expressed using a prokaryotic expression system and the purified polypeptide inhibited the growth of several tumor cell lines in vitro via apoptotic pathway. Our study revealed that CS5931 has the potential to be developed as a novel antitumor agent.     

Cloning, sequence analysis, and expression of a cDNA encoding a plastid-localized heat shock protein in maize

We have cloned and characterized a cDNA encoding a maize (Zea mays L.) heat shock protein (HSP), HSP26. The mRNA of HSP26 is present as a single mRNA species of 1.1 kilobase pairs in size and is detectable when maize seedlings are treated at 40°C but not at 28°C. Accumulation of HSP26 mRNA was detected after 10 minutes of incubation at 40°C, reaching the maximum level after 1 hour. Comparison of the deduced amino acid sequence of maize HSP26 to other HSPs indicated a strong homology to the sequences of two nuclear encoded HSPs that are transported into the chloroplasts during heat shock: pea HSP21 and soybean HSP22. Maize HSP26 was also found to cross-react with anti-pea chloroplast HSP21 antibodies. Because of the sequence homology between maize HSP26, soybean HSP22, and pea HSP21, in vitro chloroplast protein import experiments were conducted. The in vitro synthesized maize HSP26 is specifically imported to the soluble fraction of the chloroplast and processed to a smaller polypeptide. The sequence homology and antibody cross-reactivity between maize HSP26 and pea HSP21 have allowed us to conclude that maize HSP26 is a nuclear-encoded, plastid-localized protein in maize.     

粘虫核糖体蛋白S7cDNA的克隆和表达分析

运用RT—PCR和RACE技术克隆了粘虫Mythimnaseparata（Walker）核糖体蛋白s7基因（RPS7）的全长eDNA序列（GenBank登录号：JN582331）,并对其进行生物信息学分析。结果表明,粘虫RPS7全长eDNA序列为762bp,包括5’非编码区32bp和3’非编码区67bp。其开放阅读框（573bp）编码190氨基酸肽链,具有核糖蛋白S7e蛋白家族典型特征。该肽链理论分子量为21．924ku,等电点为9．82,富含4种类型的特定功能位点。该蛋白序列与其他动物RPS蛋白序列具有96．8％-98．2％高度同源性。应用荧光实时定量技术建立了粘虫脑部胚后发育RPS7表达模式。RPS7表达量随胚后发育脑部重建呈现出动态变化,这一结果显示RPS7是在转录水平上呈现发育性调节。     

粘虫核糖体蛋白S7 cDNA的克隆和表达分析

运用RT-PCR和RACE技术克隆了粘虫Mythimna separata(Walker)核糖体蛋白S7基因(RPS7)的全长cDNA序列(GenBank登录号:JN582331),并对其进行生物信息学分析。结果表明,粘虫RPS7全长cDNA序列为762 bp,包括5'非编码区32 bp和3'非编码区67 bp。其开放阅读框(573 bp)编码190氨基酸肽链,具有核糖蛋白S7e蛋白家族典型特征。该肽链理论分子量为21.924 ku,等电点为9.82,富含4种类型的特定功能位点。该蛋白序列与其他动物RPS蛋白序列具有96.8%～98.2%高度同源性。应用荧光实时定量技术建立了粘虫脑部胚后发育RPS7表达模式。RPS7表达量随胚后发育脑部重建呈现出动态变化,这一结果显示RPS7是在转录水平上呈现发育性调节。     

Cloning,characterization, and expression of cDNA encoding a lipase from Kurtzmanomyces sp. I-11

A cDNA clone of the lipase secreted by Kurtzmanomyces sp. I-11 was isolated from a cDNA library of this yeast by PCR screening using oligonucleotide primers designed on the basis of the partial amino acid sequence of the lipase. The cloned cDNA (lip1) encoded a hydrophobic protein of 484 amino acids, where the first 20 amino acids and the following 6 amino acid sequences were predicted to be the signal sequence for secretion and a pro-sequence, respectively. The deduced amino acid sequence of the Kurtzmanomyces lipase was most similar to Candida antarctica DSM 3855 lipase A (74% identity) and weakly to other lipases. The consensus pentapeptide (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases was conserved. A high level of lipase was produced by Pichia pastoris transformed with the lip1 cDNA, indicating that the cloned cDNA indeed encodes a lipase.     

Differential expression of wheat genes during cold acclimation

Overwintering crops such as winter wheat display a significant increase in freezing tolerance during periods of cold acclimation (CA). To gain a better understanding of the molecular mechanisms of CA, it is important to unravel the functions and regulations of CA-associated genes. Differential screening of a cDNA library constructed from cold acclimated crown tissue of winter wheat identified three novel CA-associated cDNA clones. Nucleotide sequence analysis showed that the clones encode a high mobility globular protein (HMGB1), a glycine-rich RNA-binding protein (TaGRP2), and a LEAD-11 dehydrin (DHN14). Accumulation of the three mRNAs during 14 days of CA was differentially regulated. In response to drought, and ABA, DHN14 mRNA rapidly accumulated while HMGB1 and TaGRP2 mRNA levels remained unchanged. The possible functions of each of these genes in cold acclimation are discussed. The text was submitted by the authors in English.     

Differential expression of wheat genes during cold acclimation

Overwintering crops such as winter wheat display significant increase in freezing tolerance during a period of cold acclimation (CA). To gain better understanding of molecular mechanisms of CA, it is important to unravel functions and regulations of CA-associated genes. Differential screening of a cDNA library constructed from cold acclimated crown tissue of winter wheat identified three novel CA-associated cDNA clones. Nucleotide sequence analysis showed that the clones encode a high mobility globular protein (HMGB1), a glycine-rich RNA-binding protein (TaGRP2), and a LEA D-11 dehydrin (DHN14). Accumulation of the three mRNAs during 14 days of CA was differentially regulated. In response to drought, and ABA, DHN14 mRNA rapidly accumulated while HMGB1 and TaGRP2 mRNA levels remained unchanged. The possible functions of each of these genes in cold acclimation are discussed.     

Cloning and characterization of a rice cDNA encoding glutamate decarboxylase

In this study, we have isolated a rice (Oryza sativa L.) glutamate decarboxylase (RicGAD) clone from a root cDNA library, using a partial Arabidopsis thaliana GAD gene as a probe. The rice root cDNA library was constructed with mRNA, which had been derived from the roots of rice seedlings subjected to phosphorus deprivation. Nucleotide sequence analysis indicated that the RicGAD clone was 1,712 bp long, and harbors a complete open reading frame of 505 amino acids. The 505 amino acid sequence deduced from this RicGAD clone exhibited 67.7 % and 61.9 % identity with OsGAD1 (AB056060) and OsGAD2 (AB056061) in the database, respectively. The 505 amino acid sequence also exhibited 62.9, 64.1, and 64.2 % identity to Arabidopsis GAD (U9937), Nicotiana tabacum GAD (AF020425), and Petunia hybrida GAD (L16797), respectively. The RicGAD was found to possess a highly conserved tryptophan residue, but lacks the lysine cluster at the C-proximal position, as well as other stretches of positively charged residues. The GAD sequence was expressed heterologously using the high copy number plasmid, pVUCH. Our activation analysis revealed that the maximal activation of the RicGAD occurred in the presence of both Ca(2+) and calmodulin. The GAD-encoded 56 approximately 58 kDa protein was identified via Western blot analysis, using an anti-GAD monoclonal antibody. The results of our RT-PCR analyses revealed that RicGAD is expressed predominantly in rice roots obtained from rice seedlings grown under phosphorus deprivation conditions, and in non-germinated brown rice, which is known to have a limited phosphorus bioavailability. These results indicate that RicGAD is a Ca(2+)/ calmodulin-dependent enzyme, and that RicGAD is expressed primarily under phosphate deprivation conditions.     

Cloning,expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis

Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent K(m) values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni(2+)-affinity column were 24.1, 6.8, 2.3, and 11.5 microM, respectively. The enzyme shows optimum activity at approximately 40 degrees C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.     

Cloning and characterization of a cDNA encoding a cowpea seed defensin and analysis of its expression

We have previously isolated a cowpea seed defensin with potent antifungal activity. Here we report the cloning of its cDNA obtained from RT-PCR. The sequence of the deduced protein VUDEF exhibited homology to plant defensins. Northern analysis revealed that VUDEF mRNA is accumulated during cowpea seed development and its level increases in seedling tissues after exposure to fungal pathogen and cold.