Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP （green fluorescent protein） fusion expression results indicate that Mtp 1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Amtpl mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.     

A Gγ subunit promoter T-DNA insertion mutant――A1-412 of Magnaporthe grisea is defective in appressorium formation, penetration and pathogenicity

Heterotrimeric G-proteins consisting of α, β and γ-subunits are essential for the transduction of ex- tracellular signals to various downstream intracellular effectors in eukaryotes. Previous studies showed that Gα and Gβ were involved in regulating     

Nucleotide sequence of cloned cDNA encoding bovine arginine vasopressin-neurophysin II precursor

The sequence of a cDNA encoding the nonapeptide arginine vasopressin (AVP) and its carrier protein, neurophysin II (NpII) from bovine hypothalamus, proves that the 166-amino acid precursor molecule contains a signal peptide of 19 amino acids followed directly by AVP connected to NpII by a Gly-Lys-Arg sequence. The carboxy-terminal region of the precursor contains a naturally occurring glycopolypeptide of 39 amino acids which is separated from NpII by a single arginine residue.     

Functional genomics in the rice blast fungus to unravel the fungal pathogenicity

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.     

Structure of the human immune interferon gene

Sequence determination of cloned cDNAs and genes of the three classes of interferon (IFN-alpha, -beta and -gamma) has revealed more than a dozen members of the human IFN-alpha gene family and a single gene for IFN-beta. These genes are found on chromosome 9 and contain no introns. We recently reported that the 146-amino acid sequence of mature IFN-gamma deduced from the nucleotide sequence of a cloned cDNA was quite unrelated to those of the other IFNs, and that the gene for IFN-gamma contains at least one intron. We now describe the isolation, characterization and DNA sequence of the human IFN-gamma gene. It contains three introns, a repetitive DNA element, and is not highly polymorphic. All our evidence to date and the present data suggest that this is the only gene for IFN-gamma and that the resolution of IFN-gamma into two components is probably the result of post-translational processing of the protein.     

Fluorescent co-localization of PTS1 and PTS2 and its application in analysis of the gene function and the peroxisomal dynamic in Magnaporthe oryzae

The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals （PTS 1 and PTS2） in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein （GFP）-PTS1, GFP-PTS2 and red fluorescence protein （RFP）-PTS1, were constructed and introduced into Magnaporthe oryzae Guy ll cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS 1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTS1 machinery was revealed by comparing the fluorescence backgrotmds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into Amgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement ofperoxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTS1 and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes.     

费尔干猪毛菜液泡膜氢离子焦磷酸酶基因SfVP的克隆、序列分析及表达

植物液泡膜氢离子焦磷酸酶(Vacuolar H+-PPase,VP)能够酸化液泡并为液泡的次级转运系统提供能量,在植物耐盐性方面发挥着着重要作用。本研究采用RT-PCR结合RACE方法,从费尔干猪毛菜中(Salsola ferganica)克隆得到液泡膜质子焦磷酸酶基因SfVP,该基因的cDNA全长2738 bp,包含1个2301 bp的完整开放读码框(ORF),编码766个氨基酸的多肽。氨基酸序列分析表明其属于H+-转运无机焦磷酸酶超家族成员,具有典型的H+-PPase结构域,其疏水性较强,含有12个跨膜螺旋结构。SfVP序列比对显示具有H+-PPase的3个保守序列CS1、CS2和CS3,其中CS1中含有保守的底物催化位点,与烟草的NtVP氨基酸序列相似性为95.3%。半定量RT-PCR结果表明,SfVP的转录表达丰度随着600 mmol/L NaCl处理时间的增加而升高,处理12 h后显著增加并一直维持在较高水平。     

内蒙古盐碱湖一株编码细菌视紫红质的嗜盐古菌bop基因全序列克隆及表达

细菌视紫红质(Bacteriorhodopsin,BR)是由bop基因编码的唯一膜蛋白,具有光推动质子泵的作用,为生命活动提供所需要的能量。为了研究高表达且具有生物活性的BR蛋白,应用PCR扩增技术,从内蒙古额吉淖尔盐碱湖分离得到的一株嗜盐古菌中扩增得到bop基因全序列;并构建了具有His6标签的原核表达载体p ET28a-IM-1,转化到大肠杆菌BL21(DE3),用IPTG诱导表达BR;并对表达产物进行SDS-PAGE鉴定分析。结果显示获得的bop基因全序列开放阅读框为672 bp,编码223个氨基酸,含有重组质粒p ET28a-IM-1的阳性菌株在IPTG诱导下高效表达了分子量约为25 k Da的BR蛋白。同源性分析表明该BR蛋白结构在古菌与细菌中具有相对较高的保守性,这与其质子泵功能密切相关。该BR蛋白为新发现的细菌视紫红质,其基因序列已递交Gen Bank,其登录号为KT873301。     

Cloning,sequencing of the HSC70 gene in Ctenopharyngodon idella

A cDNA encoding heat shock cognate protein 70(HSC70)was cloned from liver of grass carp(Ctenopharyngodon idella)(GenBank JF436930).This cDNA was found out to contain2 346 bp in length,including 1 950 bp of complete coding sequence encoding 649 amino acids(aa),plus 89 bp of 5′-UTR and 307 bp of3′-UTR.Analysis of its genomic structure revealed that its corresponding gene contained seven exons and six introns.Homology analysis indicated that it shared 99%of identity with HSC70 of breams and 86%of identity with HSP70 of Drosophila.Fluorescent RT-PCR analysis revealed that at 28℃,this gene was expressed in abdominal fat,muscle,intestines,brain,middle kidney,head kidney,gonads,swim bladder,liver,heart,spleen,gills,and fins with expression level in liver being the highest(p0.05),followed by that in the gonads;at 36℃,its mRNA expression level was increased at first but then decreased thereafter under heat shock stress,indicating that its expression can be regulated by heat shock.In conclusion,cloning and expression analysis identified a cDNA encoding a constitutive HSP70 gene that is expressed in many tissues of Ctenopharyngodon idella and its expression was down-regulated by heat shock.     

Cloning of GF14 Gene from the Bulbs of Fritillaria crrishoa

GF14 widely occurs in plant species functioning as a regulatory protein in cell signal transdution,including monoamine synthesis,exocytosis and cell cycle regulation.A partial sequence,413bp in length,is cloned from the bulbs of F.crrihosa by means of RT-CPR technique.Sequence alignment with the BLAST program shows that it is highly similar to GF14 gene.A new full-length cDNA,1133bp,is obtained by RACE techniques,It contains a major open reading frame of 262 amino acids,5‘ and 3‘untranslated regions and a long Poly(A) tail,Sequence analysis of deduced GF14 protein from F.crrihosa indicates several conserved motifs of GF14 protiens are also found in sequence of GF14 protein of F.crrihosa.     

赤红球菌甲苯胁迫响应蛋白基因的克隆、生物信息学分析和表达变化研究

从赤红球菌SD3中克隆了一个甲苯胁迫响应蛋白基因tsrp1,该基因长度为417 bp,在GenBank的登录号为MK371001.该基因编码的蛋白质Tsrp1由138个氨基酸残基组成,理论分子量为14.2 kDa,理论等电点为4.82,不稳定系数为18.68,总平均疏水指数为-0.154,属于酸性稳定的亲水蛋白.Tsrp1无信号肽和二硫键、无跨膜区、无保守结构域,存在于细胞质中.在R.ruber SD3中Tsrp1蛋白与R. rhodochrous、R. zopfii、R. pyridinivorans、R. hoagii、M. brevis和C. manganitolerans中的序列相似蛋白的相似性分别为94.93%、71.29%、67.96%、44.58%、42.05% 和35.00%.通过分子对接发现Tsrp1与c-di-GMP存在相互作用.在甲苯和苯酚胁迫下,在赤红球菌SD3中tsrp1基因的表达分别为原来的40.19倍和14.73倍.     

Fine mapping of cisc(t), a gene for cold-induced seedling chlorosis, and identification of its candidate in rice

The seedlings of indica rice cultivar Dular are susceptible to chlorosis under low temperature conditions. Our previous studies indicated that low temperature-induced seedling chlorosis is controlled by a recessive gene, located between SSR markers RM257 and RM242, on the long arm of chromosome 9. We temporarily named the gene cisc(t). Using a large F₂ population derived from a cross between Dular and the japonica cultivar Lemont, which displays a normal green color at low temperatures, cisc(t) was fine mapped to within a 12-kb interval. There is only one annotated gene in this interval, which encodes a pentatricopeptide repeat (PPR) protein. Sequence analysis indicated that 8 bases were deleted at the 60th base in the Dular allele, resulting in a frame-shift mutation and loss of function of the gene. This is consistent with the chlorosis mutant phenotype of Dular. In addition, previous studies have shown that many chlorosis mutants of seedlings are related to PPR proteins. Hence, we presume that the PPR gene is the candidate for cisc(t).     

Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously expressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridization analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium, All these results suggest PCRT might be involved in Ca^2＋ transport and storage during oyster biomineralization.     

Cloning and characterization of a gene encoding cysteine proteases from senescent leaves of Gossypium hirsutum

A gene encoding a cysteine proteinase was isolated from senescent leave of cotton (Gossypium hirsutum) cv liaomian No. 9 by utilizing rapid amplification of cDNA end spolymerase chain reaction (RACE-PCR), and a set of consensus oligonucleotide primers was designed to anneal the conserved sequences of plant cysteine protease genes. The cDNA, which designated Ghcysp gene, contained 1368 bp terminating in a poly(A)^ trail, and included a putative 5‘(98 bp) and a 3‘(235 bp) non-coding region. The opening reading frame (ORF) encodes polypeptide 344 amino acids with the predicted molecular mass of 37.88 kD and theoretical pl of 4.80. A comparison of the deduced amino acid sequence with the sequence in the GenBank database has shown considerable sequence similarity to a novel family of plant cysteine proteases. This putative cotton Ghcysp protein shows from 67% to 82% identity to the other plants. All of them share catalytic triad of residues, which are highly conserved in three regions. Hydropaths analysis of the amino acid sequence shows that the Ghcysp is a potential membrane protein and localizes to the vacuole, which has a transmembrane helix between resides 7-25. A characteristic feature of Ghcysp is the presence of a putative vacuole-targeting signal peptide of 19-amino acid residues at the N-terminal region. The expression of Ghcysp gene was determined using northern blot analysis. The Ghcysp mRNA levels are high in development senescent leaf but below the limit of detection in senescent root, hypocotyl, faded flower, 6 d post anthesis ovule, and young leaf.     

Subcellular localization and functional analyses of structural domains of COP1 in transgenic tobacco

Plants have evolved an extremely exquisite light signal regulatory network to adapt to the changing ambient light conditions, in which COP1 plays a critical role of the light signal transduction. Based on the cloned pea COP1 cDNA sequence and its protein structure, four individual gene fragments encoding different structural domains of the COP1 were designed to fuse to the GFP gene. The plant expression vectors containing these fusion genes as well as the COP1GFP fusion gene were constructed and used to transform tobacco by Agribacterium as confirmed by Southern analyses. Antibodies were raised against the recombinant GFP-COP1 overproduced in Escherichia coli. Immunoblotting results demonstrated that all of the fusion genes were constitutively expressed in transgenic tobacco plants. We systematically investigated the different subcellular localization of these fusion proteins and the resulting phenotypic characteristics of these transgenic plants under light and dark conditions. Our data show that (1) the molecular mass of the tobacco endogenous COP1 protein is 76 kD. It is constitutively expressed in all of the tested tissues and the total cellular content of COP1 protein is not noticeably affected by light conditions. (2) The nuclear localization signal of COP1 plays a critical role in regulation of its nuclear-cytoplasmic partitioning. The subcellular localization of the COP1 protein containing nuclear localization signal is regulated by light in the epidermal cells of leaves, but, it is located in nucleus constitutively in root cells. (3) The coiled-coil domain is very critical to the function of COP1 protein, while the zinc binding RING finger domain only plays a supportive role. (4) The WD-40 repeats domain is essential to the COP1 function, but this domain alone does not affect photomorphogenesis. (5) Overexpression of COP1 protein not only inhibits the photomorphogenesis of the stems and leaves of the transgenic tobacco, but also results in the generation of short and clustered roots. In contrast, overexpression of COP1 protein without WD-40 repeats domain promotes the photomorphogenesis process in the stems and leaves and lead to root elongation and lack of lateral roots. The COP1-COP1 interaction happens not only in the nucleus, but also in cytoplasm.     

Cloning, sequencing and function ofsanB, a gene related to nikkomycin biosynthesis ofStreptomyces ansochromogenes

A 6.0 kb DNA fragment related to nikkomycin biosynthesis was cloned from nikkomycin-producingStreptomyces ansochromogenes 7100. Sequence analysis showed that the 1.9 kbTth111 I fragment, a part of the 6.0 kb DNA fragment, contains one complete ORF designatedsanB (GenBank accession No. AF224501), which is composed of 1740 bp encoding a protein consisting of 580 amino acid residues. Its start codon is GTG at 100 bp position and stop codon is TGA at 1840-bp position. Database searching indicated that the deduced protein ofsanB is homologous to the histidinol-phosphate aminotransferase inStreptomyces coelicolor with 31% identities and 47% positives. Gene disruption was performed to study the function ofsanB. It was found that disruptants ofsanB lost the ability to synthesize nikkomycin, which reveals thatsanB is a novel gene essential for nikkomycin biosynthesis.