DNA sequence : the journal of DNA sequencing and mapping最新文献

Potassium is an essential element for plant, and high-affinity K+ uptake system plays a crucial role in potassium absorption and transportation. Here we report the isolation and characterization of a HKT1 homolog from C3 halophyte Suaeda salsa (L.) (SsHKT1), particularly under low K+ treatment. The SsHKT1 cDNA was 2033 nucleotides long including 1650 bp ORF for a 550 amino acids peptide and a predicted molecular mass of 63.0 kDa. The deduced amino acid sequence of SsHKT1 was 39-64% identical to other plant HKT-like sequences. A SsHKT1-specific antibody was prepared and reacted with a 63.0 kDa protein from S. salsa plasma membrane. Reverse transcriptase-PCR analysis showed that SsHKT1 was mainly expressed in leaf tissues and to a lesser extent, in root tissues. Amounts of SsHKT1 transcript were developmentally controlled and significantly up-regulated by K+ deprivation and NaCl treatment. The results suggested that SsHKT1 might play an important role in ion homeostasis and salt tolerance of S. salsa.

There are four LhcII genes of Dunaliella salina have been submitted to the database of GenBank. However, little is known about Lhca genes of this green alga, although this knowledge might be available to study the composition and phylogenesis of Lhc gene family. Recently, one Lhca gene was been cloned from the green alga D. salina by PCR amplification using degenerate primers. This cDNA, designated as DsLhca1, contains an open reading frame encoded a protein of 222 amino acids with a calculated molecular mass of 27.8 kDa. DsLhca1 is predicted to contain three transmembrane domains and a N-terminal chloroplast transit peptide (cTP) with length of 33 amino acids. The genomic sequence of DsLhca1 is composed of five introns. The deduced polypeptide sequence of this gene showed a lower degree of identity (less than 30%) with LHCII proteins from D. salina. But its homology to Lhca proteins of other algae (Volvox carteri Lhca_AF110786) was higher with pairwise identities of up to 67.1%. Phylogenetic analysis indicated that DsLhcal protein cannot be assigned to any types of Lhca proteins in higher plants or in Chlamydomonas reinhardtii.

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.

Recent year some members of mammalian chitinases and chitinase-like proteins have been discovered, but rat counterpart of human and mouse chitotriosidase has not been identified. Moreover, the physiological functions of mammalian chitinases are not very clear. To facilitate the studies we cloned the cDNA encodes the rat chitotriosidase. The results revealed that it is differ from mouse and human chitotriosidase genes, it exist alternative splicing transcripts in several tissues we detected due to different transcriptional initiation sites and different exon usage, although all the open reading frame of these cDNAs predict a protein of 464 amino acids with a typical chitinase structure, including a signal peptide, a highly conserved catalytical domain and a chitin-binding structure. The predicted amino acid sequence is highly homologous to that of mouse and human chitotriosidase. Recombinant expression of the cloned cDNA demonstrated that the encoded protein is secreted extracellularly and has chitinolytic activity.

A full-length cDNA clone with high homology to insect transferrin genes was cloned by screening a Protaetia brevitarsis cDNA library. This gene (PbTf) had a total length of 2338 bp with an open reading frame (ORF) of 2163 bp, and encoded a predicted peptide of 721 amino acid residues. Like known cockroach, termite, and beetle transferrins, PbTf appears to have residues comprising iron-binding sites in both N- and C-terminal lobes. The deduced amino acid sequence of the PbTf cDNA was closest in structure to the beetle Apriona germari transferrin (68% protein sequence identity). Northern blot analysis revealed that PbTf exhibited fat body-specific expression and was upregulated by wounding, bacterial or fungal infection and iron overload, suggesting a functional role for PbTf in defense and stress responses.

The infection-related expression of a Heterobasidion annosum (Fr.) Bref. sensu lato (s.l.) putative cytochrome P450 gene (CPM2) was analysed with realtime quantitative PCR. CPM2 was highly expressed after 20 days of growth in bark of living spruce trees, and up-regulated by nitrogen starvation on artificial media. Infection of pine seedlings in the presence of high-carbon medium results in low expression levels of CPM2, thus indicating that starvation is the primary regulatory factor for induction of this gene. The predicted cpm2 protein contains 507 amino acids with an estimated molecular mass of 56.1 kDa, and display all conserved amino acids of the cytochrome P450 protein family. The protein has a high similarity to the ord1/ordA O-methylsterigmatocystin oxidoreductases from Aspergillus flavus/A. parasiticus, responsible for catalysing the final step in aflatoxin biosynthesis. Results indicate that cpm2 is potentially important for pathogenicity in H. annosum s.l.

This is the first report of a tetraspanin (TSP)-like molecule in the lymphatic filarial parasites. Expressed sequence tag (EST) database search for TSP like molecules in the filarial genome resulted in three significant EST hits (two partial ESTs from Brugia malayi and one full length EST from Wuchereria bancrofti). The full length gene cloned from B. malayi showed significant similarity to Caenorhabditis elegans TSP and human TSP and hence the gene was named B. malayi TSP (BmTSP). Subsequent Genbank analysis with the predicted ORF of BmTSP showed additional homologous genes reported from Schistosoma mansoni and Taenia solium parasites. Structural analyses showed that BmTSP has four transmembrane domains and other conserved domains such as CCG and two other critical cysteine residues present within the large extracellular loop similar to other reported TSPs. In addition, putative post-translational modifications such as N-glycosylation, protein kinase c phosphorylation, casein kinase II phosphorylation and N-myristoylation sites have been found in BmTSP sequence. Further, PCR analyses showed that BmTSP is differentially transcribed, with highest level of expression being present in the adult stages followed by L3 and mf stages. This study thus describes a novel TSP cloned from B. malayi, its putative functions in cuticle biogenesis and role in protective immunity.

A cDNA encoding a putative H+-translocating pyrophosphatase (H+-PPase) has been cloned from Zea mays by suppression subtractive hybridization (SSH) coupled with in silico cloning approach. The isolated 2974 bp full-length cDNA named ZmGPP contains a single 2400 bp open reading frame encoding a putative protein of 799 amino acids. The predicted protein has 16 transmembrane domains and is significantly similar to Golgi apparatus resident type-II H+-PPase from Arabidopsis thaliana. DNA gel blotting analysis shows that ZmGPP is a low-copy gene. Organ expression pattern analysis reveals that ZmGPPexpressed highly in leaf and tassel, followed by in stem, root, and ear. The Real-time RT-PCR assays showed that the expression of ZmGPP was up-regulated both in shoots and roots of maize seedlings under dehydration, cold and high salt stresses. Those results suggest that the ZmGPP product may play an important role in abiotic stress tolerance of Z. mays.

Cystatins are physiological cysteine proteinase inhibitors. We used digital differential display (DDD) to clone two novel splice variants Rcet1-v1 and Rcet1-v2 which were isolated from adult mouse testis cDNA library. Sequence analysis revealed that Rcet1-v1 and Rcet1-v2 cDNAs are 454 and 610 bp in length, respectively, and each has four exons, but the lengths of their second and third exons are different, with the results that these cDNAs encoded two different putative proteins. The deduced proteins were 88 amino acid residues (RCET1-v1) and 140 residues (RCET1-v2) in length and have one potential signal peptide and one cystatin domain, respectively, but lack part critical consensus sites important for cysteine protease inhibition. These characteristics are seen in CRES subgroup, which related to the family 2 cystatins and primarily expressed in reproductive tract. RT-PCR analysis showed that Rcet1-v1 and Rcet1-v2 were specifically expressed in adult mouse testis, epididymis and cerebrum, but higher in testis than in epididymis and cerebrum. RT-PCR analysis also showed that Rcet1-v1 and Rcet1-v2 were specifically expressed in adult mouse pituitary and spermatogonium, but not expressed in spermatozoa. Results of in situ hybridization showed that Rcet1 gene expressed abundantly in mouse spermatogonium, spermatocytes and round spermatids; did not expressed in spermatozoa. At mouse testis different development stages, Rcet1-v1 and Rcet1-v2 were expressed very low from postnatal 1 day to postnatal 3 weeks; after postnatal 4 weeks, expressed steadily increased from postnatal 4 to 7 weeks, highest in postnatal 7 to 8 weeks, then keeping on the expressing level of postnatal 6 weeks in postnatal 13-57 weeks. All these indicated that Rcet1-v1 and Rcet1-v2 primarily expressed in mouse male reproductive tract and may play important roles in mouse spermatocytes and round spermatid development. Rcet1-v1 and Rcet1-v2 may be new members of Cres subgroup of the family 2 cystatins.

The product of the POUSF1 gene, Oct4, plays an important role both in embryonic development and in the self-renewal and differentiation of totipotent cells. To understand the function of Oct4 in rabbit ES cells, we cloned and sequenced the rabbit POU5F1 gene, as well as the cDNA encoded by the gene. The Oct4 cDNA contains a 1083 bp ORF encoding a 360 aa protein and a 241 bp 3' UTR sequence. Oct4 mRNA was expressed at a high level in rabbit ES cells and was barely detectable in the adult spleen, kidney, brain and muscle tissues. The POU5F1 gene is approximately 6 kb in length and includes five exons and four introns. Gene organization is similar to that of the mouse, human and bovine orthologs. Sequencing of the gene revealed an 82% (mouse), 90% (human) and 89% (bovine) overall identity at the protein level. The rabbit POUSF1 gene was mapped to chromosome 12q1.1 by PCR amplification of DNA from two putative POU5F1-containing BAC clones, which were previously mapped to chromosome 12q1.1. The cloning of the rabbit POU5F1 gene will facilitate studies on its roles in rabbit embryogenesis and ES cells.