水稻组蛋白H_3基因的分离和组织结构分析

用噬菌斑原位杂交法,从一个水稻新品系的EMBL,基因文库中筛选到了两个组蛋白H,基因克隆,并制作了它们的限制性内切酶图谱,分析了它们的组织结构。其中λRH_3-1克隆包含2个组蛋白H_3基因区(分别为0.5kb和0.8kb的BamHI-EcoRI片段,两基因区距离5.8kb)。而λRH_3-2克隆只带有1个结构特异的H_3基因区(5.2kb的EcoRI-HindⅢ片段)。本文还就水稻组蛋白H_3基因在整个基因组中的组织结构特点进行了讨论。     

水稻中一个新的MYC基因的克隆及其分析

在水稻基因组序列中发现类似MYC序列的同源序列,并设计引物成功克隆了一个水稻OsMYC基因(GenBank登录号：AY398581),并对其进行了分子结构分析。OsMYC基因具有典型的DNA结合结构域：碱性区域／螺旋-环-螺旋(bHLH)基序,与其他MYC类似基因的蛋白序列比对结果表明,OsMYC基因与AtMYC2、MYC7E和PG1等氨基酸序列一致性分别是78%、48％、46％,而在bHLH区的一致性分别为95％、84％、77％,N端保守区的一致性分别为81％、54％、52％;位于bHLH区域内的核定位信号区则完全一致;系统进化树分析结果表明,该基因与AtMYC2、MYC7E和PG1等位于同一亚类。此外,该基因主要在营养器官中表达,茎秆中表达最强,根和叶片中较弱,并且能被外源ABA或Fe^3 所诱导,这与AtMYC2、MYC7E基因的表达模式基本相同。该基因是水稻MYC转录因子家族的一个新成员。     

Molecular Characterization of a Genomic Fragment Containing Pi-kh Gene from the Genomic Library of Indica Rice Line Tetep

Identification of full length genes along with upstream regulatory elements is important to understand its expression. Here, we report preparation of high titre genomic library and identification of a genomic clone containing Pi-k ^h gene with its complete upstream and downstream sequences from the rice blast resistant line Tetep. Structural analysis of protein revealed that Pi-k ^h has a central nucleotide binding site domain, leucine-rich repeats domain and a unique zinc-finger domain. Comparative analysis of Pi-k ^h protein sequence showed 64% and 45% similarity with the protein sequences of rice blast resistance genes Pi-b and Pi-ta , respectively.     

Isolation and Characterization of a Rice Cysteine Protease Gene, OsCP1, Using T-DNA Gene-Trap System

The T-DNA gene-trap system has been efficiently used to elucidate gene functions in plants. We report here a functional analysis of a cysteine protease gene, OsCP1, isolated from a pool of T-DNA insertional rice. GUS assay with the T-DNA tagged line indicated that the OsCP1 promoter was highly active in the rice anther. Sequence analysis revealed that the deduced amino acid sequence of OsCP1 was homologous to those of papain family cysteine proteases containing the highly conserved interspersed amino acid motif, ERFNIN. This result suggested that the gene encodes a cysteine protease in rice. We also identified a suppressed mutant from T2 progeny of the T-DNA tagged line. The mutant showed a significant defect in pollen development. Taken together, the results demonstrated that OsCP1 is a cysteine protease gene that might play an important role in pollen development.     

Isolation and Characterization of a Ferredoxin Gene from Arabidopsis thaliana

We report the cloning and characterization of an Arabidopsis thaliana (L.) Heynh. (Columbia ecotype) ferredoxin gene (Fed A). Sequence analysis of a genomic clone shows an intron-free, 444-base pair open reading frame which encodes a 96 amino acid mature ferredoxin polypeptide preceded by a 52 amino acid transit peptide. Comparison with other plant ferredoxin proteins suggests that Fed A encodes a leaf ferredoxin. Genomic Southern blot analysis indicates the presence of a second, weakly related gene, consistent with other reports of at least two ferredoxins in plants. The Fed A gene promoter contains two regions, ACGCCACGTGGTAGATAGGATT (G-I box) and CCACGCCATTTCCACAAGC (CCAC box), which are strongly conserved in both sequence and position between the Arabidopsis and pea ferredoxin genes. Similarities with other better characterized plant promoter elements are also discussed.     

Isolation and Characterization of a Chitinase and its cDNA Clone from Rice

A 35 kD chitinase has been purified to apparent homogeneity from extracts of rice bran of cv New Bonnet by ammonium sulfate fractionation, chitin affinity chromatography, cation exchange chromatography on carboxymethyl cellulose and gel filtration. The purified enzyme has an isoelectric point of 8.8. The enzyme inhibited the growth of Rhizoctonia solani (the sheath blight pathogen), Trichoderma viride, T. harzianum, Fusarium graminaerum and F. culmorum in vitro. A cDNA clone for chitinase was isolated from a developing rice seed cDNA library by probing with a barley chitinase cDNA probe. The nucleotide sequence of this 654 bp clone was determined, it contains an open reading frame of 519 nucleotides. The protein product encoded by this clone is homologous to chitinases from tobacco, bean and barley. Southern blot analysis of rice genomic DNA with this probe revealed that chitinases are encoded by a small multi-gene family in the rice genome.     

The Rice B-Box Zinc Finger Gene Family: Genomic Identification,Characterization, Expression Profiling and Diurnal Analysis

Background

The B-box (BBX) -containing proteins are a class of zinc finger proteins that contain one or two B-box domains and play important roles in plant growth and development. The Arabidopsis BBX gene family has recently been re-identified and renamed. However, there has not been a genome-wide survey of the rice BBX (OsBBX) gene family until now.

Methodology/Principal Findings

In this study, we identified 30 rice BBX genes through a comprehensive bioinformatics analysis. Each gene was assigned a uniform nomenclature. We described the chromosome localizations, gene structures, protein domains, phylogenetic relationship, whole life-cycle expression profile and diurnal expression patterns of the OsBBX family members. Based on the phylogeny and domain constitution, the OsBBX gene family was classified into five subfamilies. The gene duplication analysis revealed that only chromosomal segmental duplication contributed to the expansion of the OsBBX gene family. The expression profile of the OsBBX genes was analyzed by Affymetrix GeneChip microarrays throughout the entire life-cycle of rice cultivar Zhenshan 97 (ZS97). In addition, microarray analysis was performed to obtain the expression patterns of these genes under light/dark conditions and after three phytohormone treatments. This analysis revealed that the expression patterns of the OsBBX genes could be classified into eight groups. Eight genes were regulated under the light/dark treatments, and eleven genes showed differential expression under at least one phytohormone treatment. Moreover, we verified the diurnal expression of the OsBBX genes using the data obtained from the Diurnal Project and qPCR analysis, and the results indicated that many of these genes had a diurnal expression pattern.

Conclusions/Significance

The combination of the genome-wide identification and the expression and diurnal analysis of the OsBBX gene family should facilitate additional functional studies of the OsBBX genes.     

水稻OsGSTL1启动子的分离和表达分析

从水稻基因组文库中筛选得到一个水稻GST基因,命名为OsGSTL1.半定量RT-PCR分析表明OsGSTL1基因的表达不受绿磺隆、乙烯利、脱落酸、水杨酸和茉莉酸甲酯的诱导,因此该基因可能与植物抗逆性无关.为了研究OsGSTL1启动子在植物体内的表达特性,将OsGSTL1起始位点5＇端上游不同长度的调控序列与报告基因GUS融合,并在洋葱表皮瞬间表达和拟南芥中稳定表达.研究表明：在洋葱表皮细胞中,160bp及更长的上游调控序列均能启动GUS基因的表达;而在转基因拟南芥中,含有2155 bp的上游序列的PGZ2.1：：GUS具有时空表达的特性,在转基因的早期幼苗中GUS基因在子叶中特异性表达,但在根中没有表达;而在幼苗生长的后期,根、茎、叶中都有少量的表达.但包含1 224 bp的上游序列的PGZ1.2：：GUS却表现为组成型表达的特性.由此推测,OsGSTL1启动子启动的基因表达可能与幼苗的营养代谢相关;而OsGSTL1启动子的时空表达相关元件可能位于OsGSTL1翻译起始位点5＇端上游-2155 bp至-1224 bp范围内.     

A Chitinase Encoding Gene (chit1 Gene) from the Entomopathogen Metarhizium anisopliae: Isolation and Characterization of Genomic and Full-Length cDNA

There are no reports to date of entire gene sequences coding for chitinolytic enzymes from entomopathogenic fungi, even though these enzymes act synergistically with proteolytic enzymes to solubilize insect cuticle during the key step of host penetration, having considerable importance in the biological control of some insect pests. This paper reports the complete nucleotide sequence and analysis of the chromosomal and full-length cDNA copies of the regulated gene (chit1) coding one of the chitinases produced by the biocontrol agent Metarhizium anisopliae. Degenerated primers, encompassing conserved regions of other fungal chitinases, were used to amplify a 650-bp DNA fragment, which was used to isolate genomic and cDNA clones from M. anisopliae. Albeit at least two different chitinases are characterized in this fungus, only one chit gene was isolated. The chit1 gene is interrupted by three short typical fungal introns and has a 1,521-bp ORF, which encodes a protein of 423 amino acids with a stretch of 35 amino acid residues displaying characteristics of signal peptide. The deduced sequence of the mature protein predicts a 42-kDa protein with pI of 5.8. Southern analysis of genomic DNA indicates a single copy of chit1 in the M. anisopliae genome. Received: 13 March 1998 / Accepted: 14 April 1998     

Isolation and Characterization of a Heme Oxygenase-1 Gene from Chinese Cabbage

Heme oxygenase-1 (HO1) is a heme-catabolizing enzyme induced by a variety of stress conditions. This article described the cloning and characterization of BrHO1 gene which codes for a putative HO1 from Chinese cabbage (Brassica rapa subsp. pekinensis). BrHO1 consists of three exons and encodes a protein precursor of 32.3 kD with a putative N-terminal plastid transit peptide. The amino acid sequence of BrHO1 was 84% similar to Arabidopsis counterpart HY1. The three-dimensional structure of BrHO1 showed a high degree of structural conservation compared with the known HO1 crystal structures. Phylogenetic analysis revealed that BrHO1 clearly grouped with the HO1-like sequences. The recombinant BrHO1 protein expressed in Escherichia coli was active in the conversion of heme to biliverdin IXα (BV). Furthermore, the results of subcellular localization of BrHO1 demonstrated that BrHO1 gene product was most likely localized in the chloroplasts. BrHO1 was differently expressed in all tested tissues and could be induced upon osmotic and salinity stresses, cadmium (Cd) exposure, hydrogen peroxide (H₂O₂), and hemin treatments. Together, the results suggested that BrHO1 plays an important role in abiotic stress responses.     

Isolation and Characterization of a Cytokinin Up-Regulated Gene from Tobacco Mesophyll Protoplasts

We have isolated a cytokinin up-regulated cDNA clone, H13, froman early stage of cultured tobacco mesophyll protoplasts bya differential display method. The expression of this gene wasspecifically induced by natural and synthetic cytokinins includingN-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30), a diphenylurea-typecytokinin, although the simultaneous presence of auxin was alsorequired. It seems that the preceding treatment of the tobaccomesophyll protoplasts by auxin is necessary for the gene torespond to cytokinin. The addition of a cytokinin antagonist,compound 182, which suppressed the induction of cell divisionin tobacco mesophyll protoplasts, completely abolished the expressionof this gene. Though the predicted gene product of H13 did notsuggest us any sequences of defined functions, two domains ofthe predicted sequence had significant homology to several reportedsequences in the data base. The gene product of H13 is proposedto have a role in regenerating cell wall in cultured protoplasts,since a cDNA clone E6, from cotton fiber cells, which has themost closely related structure to H13, has been isolated fromcells which showed active cellulose synthesis. This suppositionis supported by the evidence that in the absence of cytokinin,cell wall regeneration was significantly suppressed, resultingin failure of the induction of cell division. Thus, the geneproduct of H13 is supposed to have a role in regenerating cellwalls and facilitating the progression of the cell cycle, resultingin the sustained cell division of tobacco mesophyll protoplasts. ¹These authors are equally contributed to this work.     

Isolation and Characterization of a Putative Class E Gene from Taihangia rupestris

Studies In model plants showed that SEPALLATA （SEP） genes are required for the Identification of floral organs and the determination of floral meristems In Arabidopsis. In this paper a SEP homolog, TrSEP3, was Isolated from a China-specific species, Taihangla rupestrisi Yü et LI. Phylogenetlc analysis showed that the gene belongs to the SEP3-clade of SEP （previous AGL2） subfamily. In situ hybridization was used to reveal the potential functional specification, and the results showed that TrSEP3 expression was first observed in floral meristems and then confined to the floral primordla of the three inner whorls. In the matured flower, TrSEP3 was strongly expressed In the tips of pistils and weak In stamens and petals. The evolution force analysis shows that TrSEP3 might undergo a relaxed negative selection. These results suggested that TrSEP3 may not only function In determining the identity of floral merlstems and the primordia of three inner whorls, but also function In matured reproductive organs.     

Isolation, Characterization, Molecular Gene Cloning, and Sequencing of a Novel Phytase from Bacillus subtilis

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55°C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.     

Isolation and Genomic Characterization of a Duck-Origin GPV-Related Parvovirus from Cherry Valley Ducklings in China

A newly emerged duck parvovirus, which causes beak atrophy and dwarfism syndrome (BADS) in Cherry Valley ducks, has appeared in Northern China since March 2015. To explore the genetic diversity among waterfowl parvovirus isolates, the complete genome of an identified isolate designated SDLC01 was sequenced and analyzed in the present study. Genomic sequence analysis showed that SDLC01 shared 90.8%–94.6% of nucleotide identity with goose parvovirus (GPV) isolates and 78.6%–81.6% of nucleotide identity with classical Muscovy duck parvovirus (MDPV) isolates. Phylogenetic analysis of 443 nucleotides (nt) of the fragment A showed that SDLC01 was highly similar to a mule duck isolate (strain D146/02) and close to European GPV isolates but separate from Asian GPV isolates. Analysis of the left inverted terminal repeat regions revealed that SDLC01 had two major segments deleted between positions 160–176 and 306–322 nt compared with field GPV and MDPV isolates. Phylogenetic analysis of Rep and VP1 encoded by two major open reading frames of parvoviruses revealed that SDLC01 was distinct from all GPV and MDPV isolates. The viral pathogenicity and genome characterization of SDLC01 suggest that the novel GPV (N-GPV) is the causative agent of BADS and belongs to a distinct GPV-related subgroup. Furthermore, N-GPV sequences were detected in diseased ducks by polymerase chain reaction and viral proliferation was demonstrated in duck embryos and duck embryo fibroblast cells.     

Isolation and Characterization of a Novel Human Radiosusceptibility Gene, NP95

The murine nuclear protein Np95 has been shown to underlie resistance to ionizing radiation and other DNA insults or replication arrests in embryonic stem (ES) cells. Using the databases for expressed sequenced tags and a two-step PCR procedure, we isolated human NP95, the full-length human homologue of the murine Np95 cDNA, which consists of 4,327 bp with a single open reading frame (ORF) encoding a polypeptide of 793 amino acids and 73.3% homology to Np95. The ORF of human NP95 cDNA is identical to the UHRF1 (ubiquitin-like protein containing PHD and RING domain 1). The NP95 gene, assigned to 19p13.3, consists of 18 exons, spanning 60 kb. Several stable transformants from HEK293 and WI-38 cells that had been transfected with the antisense NP95 cDNA were, like the murine Np95-knockout ES cells, more sensitive to X rays, UV light and hydroxyurea than the corresponding parental cells. In HEK293 cells, the lack of NP95 did not affect the activities of topoisomerase IIalpha, whose expression had been demonstrated to be regulated by the inverted CCAAT box binding protein of 90 kDa (ICBP90) that closely resembles NP95 in amino acid sequence and in cDNA but differs greatly in genomic organization. These findings collectively indicate that the human NP95 gene is the functional orthologue of the murine Np95 gene.     

Isolation and Characterization of IaYABBY2 Gene from Incarvillea arguta

The establishment of ad-abaxial polarity is an important characteristic of the development of lateral organs in plants. YABBY genes encode higher plant-specific nuclear proteins which play critical roles in promoting abaxial cell fate. IaYABBY2 (IaYAB2, Genbank accession no. KF250432), isolated from Incarvillea arguta, is a member of YABBY gene family. Sequence characterization and phylogenetic analyses show that IaYABBY2 is a member of the YAB2 subfamily of Arabidopsis thaliana. Subcellular localization analysis indicates that IaYABBY2 is localized in the nucleus. Ectopic expression of IaYABBY2 in Arabidopsis plants resulted in the partial abaxialization of adaxial epidermises of leaves and sepals and development defect of florescence. The transgenic lines also showed higher level of anthocyanin content and photosynthesis capability after differential environment stress. These results indicate that the IaYABBY2 functions in the ad-abaxial polarity, development of shoot apical meristem, and environmental stress.