A cDNA encoding a putative SPF1-type DNA-binding protein from cucumber

A cDNA clone encoding a polypeptide with homology to the novel SPF1 DNA-binding protein of sweet potato has been isolated from a cDNA library from RNA of senescing cucumber (Cucumis sativus, L.) cotyledons. Comparison of the two sequences reveals similar features which may be important in the evolution and function of this protein, including a duplicated region of about 56 amino acids (aa). The first half of the duplicated region is enriched in basic aa and is very highly conserved, both within and between each polypeptide. In contrast, the second half of the duplicated region is poorly conserved within each polypeptide, but highly conserved when cucumber and sweet potato sequences are compared. Southern blot analysis with cucumber DNA shows a simple hybridisation pattern indicating one or very few genes. Northern blot analysis shows that the expression of the cucumber gene increases in cotyledons as they expand and become photosynthetic and remains high in senescence. The possibility that the cucumber SPF1-type protein may be involved in carbohydrate regulation of gene expression is discussed.     

Characterization of a single-copy Arabidopsis gene encoding a protein showing limited similarity to the N-terminus of the mammalian clathrin-assembly protein AP180.

The Arabidopsis 194 gene encoding a protein containing sequence similarity to an N-terminal region of the clathrin-assembly protein AP180 has been identified in a 4.9-kb region of genomic DNA upstream of the gene encoding the high mobility group protein HMG-I/Y. The gene consists of 12 exons and 11 introns, identified by comparison with partial cDNAs and using the NetPlantGene programme, and encodes a protein of 584 amino acid residues. The C-terminal region of the protein contains 8 tandem repeats of a 17-amino-acid-residue sequence. Southern blot analysis of genomic DNA from Columbia and Landsberg ecotypes of Arabidopsis indicates the presence of a single copy of the 194 gene. The 194 gene is expressed in all organs of Arabidopsis including roots, stems, leaves, flowers, and developing siliques, as determined by northern blot analysis.     

霍乱弧菌zot基因的克隆及其在大肠杆菌中的表达

从霍乱疫苗菌中抽提基因组ＤＮＡ,用ＰＣＲ的方法扩增ｚｏｔ基因。序列分析表明,ｚｏｔ基因编码３９９个氨基酸,其中４个氨基酸与文献报道有差异。将ｚｏｔ基因插入含Ｔ７启动子的质粒ｐＥＴ－２８（ａ＋）构建表达质粒ｐＥＴ－ＺＯＴ,转化大肠檑菌ＢＬ２１（ＤＥ３）筛有达菌株ＢＬＺＯＴ。表达菌株经１ｍｍｏｌ／ＬＴ ＩＰＴＧ诱导表达３－５ｈ后,表达大量ＺＯＴ蛋白,并形成包涵体,经ＳＤＳ－ＰＡＧＥ分析重组ＺＯＴ蛋白分     

Cloning, Sequence, and Expression Analysis of a New MnSOD-Encoding Gene from the Root-Knot Nematode Meloidogyne incognita

A gene encoding a manganese superoxide dismutase (MnSOD) enzyme (Mi-mnsod) was identified and characterized in second-stage juveniles of the root-knot nematode Meloidogyne incognita. The Mi-mnsod gene was found to possess five exons and four introns with (GT/AG) consensus splice-site junctions. The deduced amino acid sequence of Mi-mnsod encodes a putative 25 KDa protein, with conserved amino acid residues of the MnSOD family, including the Parker-Blake signature and four metal-binding sites. The derived amino acid sequence showed high similarity to other eukaryotic MnSODs, including a 23 amino acid N-terminal putative mitochondrial transit peptide. Gene expression was observed throughout the posterior nematode body region with elevated signal intensities at the anterior portion of the intestine. DNA blot analysis and sequencing data showed the occurrence of three putative copies of the MnSOD gene with nucleotide polymorphisms found at the fourth exon and the 3' un-translated region.     

Interaction of wheat high-mobility-group proteins with four-way-junction DNA and characterization of the structure and expression of HMGA gene

Plant high-mobility-group (HMG) chromosomal proteins are the most abundant and ubiquitous nonhistone proteins found in the nuclei of higher eukaryotes. There are only two families of HMG proteins, namely, HMGA and HMGB in plants. The cDNA encoding wheat HMGa protein was isolated and characterized. Wheat HMGA cDNA encodes a protein of 189 amino acid residues. At its N terminus, there is a histone H1-like structure, which is a common feature of plant HMGA proteins, followed by four AT-hook motifs. Polymerase chain reaction results show that the gene contains a single intron of 134 bp. All four AT-hook motifs are encoded by the second exon. Northern blot results show that the expression of HMGA gene is much higher in organs undergoing active cell proliferation. Gel retardation analysis show that wheat HMGa, b, c and histone H1 bind to four-way-junction DNA with high binding affinity, but affinity is dramatically reduced with increasing Mg(2+) and Na(+) ion concentration. Competition binding studies show that proteins share overlapping binding sites on four-way-junction DNA. HMGd does not bind to four-way-junction DNA.     

Cytosolic triosephosphate isomerase is a single gene in rice.

A cDNA clone encoding rice (Oryza sativa L.) cytosolic triosephosphate isomerase (TPI), an important glycolytic enzyme, was isolated and characterized. The clone (pRTPI-6) contains an open reading frame of 759 base pairs, encoding a polypeptide chain of 253 amino acid residues (M(r) 27,060). The identity of this clone was defined by its high homology (85% nucleotide sequence and 89% amino acid sequence identical match) with a maize mRNA sequence encoding the cytosolic TPI and with TPIs from other species. Genomic DNA blot analysis using the cDNA as a probe showed that the cytosolic TPI gene is present as a single copy per haploid rice genome, as opposed to that found in maize, in which multiple TPI gene copies exist. A single TPI mRNA species of about 1100 nucleotides was detected by gel blot hybridization analysis of RNA isolated from root, culm, and leaf tissues, indicating that its expression is ubiquitous. Based on sequence comparison and molecular analysis, we propose that the chloroplast-located TPI may be encoded by divergent structural nuclear genes in rice.     

A Serine Protease Gene from the Firefly, <Emphasis Type="Italic">Pyrocoelia rufa</Emphasis>: Gene Structure,Expression, and Enzyme Activity

The gene structure, expression and enzyme activity of a serine protease from the firefly, Pyrocoelia rufa (PrSP) were examined. The PrSP gene spans 1474 bp and consists of two introns and three exons coding for 257 amino acid residues. Southern blot analysis of genomic DNA suggested the presence of PrSP gene as a single copy. Western blot analysis and enzyme activity assay exhibited midgut-specific expression, suggesting that the midgut is the prime site where large quantities of PrSP are synthesized for degrading the absorbed protein from the diet. The cDNA encoding PrSP was expressed as a 31 kDa polypeptide in the baculovirus-infected insect Sf9 cells and the recombinant PrSP showed activity in the protease enzyme assay using gelatin as a substrate.     

Isolation and characterization of a dehydrin gene from Cicer pinnatifidum, a drought-resistant wild relative of chickpea

Chickpea is an important legume crop of the tropics and subtropics. As it is cultivated mostly during the dry season, drought is the major cause of poor chickpea yields. A wild relative of chickpea, Cicer pinnatifidum , is more tolerant than chickpea itself to various abiotic stresses, including drought. A cDNA clone encoding a dehydrin gene, cpdhn1 , was isolated from a cDNA bank prepared from ripening seeds of C. pinnatifidum . Dehydrins are proteins which accumulate in seeds during late embryogenesis and also during stress brought about by drought and other abiotic factors in many plants. The polypeptide deduced to correspond to this gene, cpdhn1 , consists of 195 amino acid residues with a molecular mass of 20.4 kDa. The polypeptide is a Y₂K dehydrin exhibiting two conserved 'Y'-segments in the amino-terminal region and one conserved 'K'-segment close to the carboxy terminus. Northern blot analyses showed that cpdhn1 expression was induced not only during seed development, but also in leaves in response to drought, chilling and salinity and also to treatment with ABA or methyl jasmonate. The induction of cpdhn1 expression by methyl jasmonate and ABA indicates that the gene may also be involved in the response to biotic stress. The CpDHN1 protein may thus improve the tolerance of chickpea to a variety of environmental stresses, both abiotic and biotic.     

茶树CsGME1基因的克隆与逆境胁迫响应分析

该研究基于茶树转录组和基因组信息,以茶树‘龙井43’为实验材料,从其cDNA中克隆获得茶树CsGME1基因,并对其蛋白序列特征、基因表达模式及其在不同非生物胁迫下的表达水平进行实时荧光定量分析。结果显示:(1)茶树CsGME1开放阅读框长度为1131 bp,编码376个氨基酸;该序列与多个相关物种的GME氨基酸序列一致性为94.25%,均含有NAD结合域。(2)进化树分析表明,茶树CsGME1基因与番茄SlGME1亲缘关系较近,与水稻OsGME2亲缘关系最远。(3)CsGME1蛋白属于亲水性蛋白,理论相对分子量为42046.84 Da,理论等电点为5.73,具有4个无序化区域,无序化程度较低;CsGME1蛋白二级结构由39.25%α-螺旋,13.26%延伸主链,5.84%β-转角和41.38%随机卷曲组成;三级结构分析结果显示,CsGME1包含螺旋和随机卷曲,与二级结构吻合。(4)荧光定量分析结果显示,在高温(38℃)、低温(4℃)、干旱(20%PEG)和高盐(200 mmol·L-1 NaCl)4种非生物胁迫处理下,茶树CsGME1基因均有响应,且表达存在差异,推测CsGME1参与了茶树的逆境胁迫响应过程。     

Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.     

Suppressed expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in an irradiation-attenuated Plasmodium berghei XAT strain

Plasmodium berghei XAT (XAT) is a non-reversible, non-lethal type malaria parasite strain derived from the highly virulent lethal P. berghei NK65 (NK65) by X-irradiation. The difference in polypeptide expression between NK65 and XAT was examined in this study. Western blot patterns of the parasite polypeptides showed that a 30-kDa polypeptide was not detected in XAT. In the present paper, we focused the study on the difference in the expression of the 30-kDa polypeptide between XAT and NK65. Although several other significant differences were noted in the spots shown by two-dimensional gel electrophoresis, the 30-kDa polypeptide was isolated by means of preparative 2D-gel electrophoresis followed by HPLC, and N-terminal amino acid sequence of the polypeptide was eventually determined. Complementary DNA clones encoding the 30-kDa polypeptide were isolated and characterized. Full-length cDNA clones from XAT encoded a protein of 231 amino acid residues with a 693-bp open reading frame. The deduced amino acid sequence exhibited 67% identity with that for P. falciparum hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8), suggesting that this protein is P. berghei HGPRT. Northern blot analysis revealed that expression of HGPRT in XAT was only one-eighth of that in NK65. This finding indicates that HGPRT gene expression is markedly suppressed in XAT. The amino acid sequence of HGPRT from NK65 was identical to that from XAT. This finding showed that the amino acid sequence of XAT-HGPRT was not mutated and had not undergone deletion.     

A proline-rich chitinase from Beta vulgaris

A gene (Chl) encoding a novel type of chitinase was isolated from Beta vulgaris. The Ch1 protein consists of an N-terminal hydrophobic prepeptide of 25 amino acids followed by a hevein-like domain of 22 amino acid residues, an unusually long proline-rich domain of 131 amino acid residues with 90 prolines, and finally a catalytic domain of 261 amino acid residues. Proteins with similar proline-rich domains are present in some other plants. The Chl gene shows a transient expression in response to fungal infection.