Embryo and anther regulation of the mabinlin II sweet protein gene in Capparis masaikai Lévl

Mabinlin II is one of the major sweet proteins stored in the seeds of Capparis masaikai Lévl. Its promoter region (779 bp) located 5′ upstream of the mabinlin II gene has been isolated and named as MBL-779 (GenBank accession number, EU014073). This promoter contains two typical TATA box regions and a series of motifs related to seed-specific promoters, such as ACGT motifs, RY motif, napin motif, and G box. The MBL-779 promoter drove GUS gene to transiently express in the embryos of bean, maize, and rice seeds or to constantly express in the embryos and anthers of the transgenic Arabidopsis. The MBL-779 promoter regulated gene expression from approximately the 12th day and peaked on approximately the 16th day after flowering in Arabidopsis. The −300-bp promoter region is a minimal sequence required to functionally regulate gene expression. The CAATs at −325 to −322 bp and −419 to −416 bp and the region at −485 to −770 bp play a role in the quantitative regulation of gene expression. The RY motif, CATGAC, at −117 to −112 bp and the ACGT within the G box (CACGTG) at −126 to −123 bp positively regulate gene expression. X.-W. Hu and S.-X. Liu have the same contribution as first author.     

Investigations into the taxonomy of the mushroom pathogen <Emphasis Type="Italic">Verticillium fungicola</Emphasis> and its relatives based on sequence analysis of nitrate reductase and ITS regions

The full sequence of the nitrate reductase gene was obtained from a type isolate of Verticillium fungicola var. fungicola and used for phylogenetic analysis against other ascomycete fungi. Sequencing obtained 2749 bp of coding region, 668 bp of 5′ flanking sequence and 731 bp of 3′ flanking sequence. In silico analysis indicated that the coding region contains a single intron and translates into an 893 amino acid protein, with BLAST analysis identifying five conserved nitrate reductase domains within the protein. The 5′ flanking sequence contains numerous conserved sites putatively involved in binding nitrogen regulatory proteins, indicating that the regulation of the gene is likely to be subject to the same regulation as that of model fungi such as Aspergillus nidulans. The central portion of this gene was amplified and sequenced from a number of V. fungicola isolates and related fungi and the resulting phylogenies compared to those obtained from analysis of the rDNA internal transcribed spacer regions for these fungi. Both nitrate reductase and ITS analyses provide additional evidence that reinforces previous findings that suggest the mushroom pathogenic Verticillium species are more related to other chitinolytic fungi such as the insect pathogens Verticillium lecanii and Beauveria bassiana than to the plant pathogenic Verticillia.     

Molecular characterization,chromosomal localization and association analysis with back-fat thickness of porcine <Emphasis Type="Italic">LPIN2</Emphasis> and <Emphasis Type="Italic">LPIN3</Emphasis>

The products of mammalian LPIN2 and LPIN3 are phosphatidate phosphatase type 1 enzymes, which play an important role in the de novo biosynthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine. In this study, we obtained a 2,985-bp cDNA sequence of porcine LPIN2, which contains a 2,676-bp open reading frame flanked by an 11-bp 5′UTR and a 298-bp 3′UTR, and a 2,843-bp cDNA sequence of porcine LPIN3, which contains a 111-bp 5′UTR, a 2,580-bp open reading frame and a 152-bp 3′UTR. RT-PCR analysis showed that both LPIN2 and LPIN3 mRNA were ubiquitously expressed with a very high level in liver. By using the somatic cell hybrid panel (SCHP) and the radiation hybrid (IMpRH) panel, porcine LPIN2 and LPIN3 were assigned to 6q24-(1/2)q31 and 17(1/2)q21-q23, respectively. One T2193C single nucleotide polymorphism in LPIN2 was identified and was detected by Hin6I PCR-RFLP. Association analysis showed that different genotypes of LPIN2 were associated with back-fat thickness between the 6th and 7th ribs (P < 0.01).     

A full lengthTy3/gypsy-type retrotransposon in the fernAdiantum

Ty3/gypsy-type LTR-retrotransposons have been found only in lily and maize but not in cryptogam. In fernAdiantum, we recently found a full-lengthTy3/gypsy-type LTR-retrotransposon (ARET-1; 8284 bp). This retrotransposon has both 5′ and 3′ LTRs (1.2 kb), a primer binding site, a polypurine tract, and an RNA binding motif and its domain arrangement in thepol region is the same as that ofTy3/gypsy-type retrotransposon. These results suggest thatTy3/gypsy-type retrotransposons are widespread among vascular plants. The nucleotide sequence data reported will appear in the EMBL, DDBJ and GenBank Nucleotide Sequence Databases under the accession number AB003364.     

Major histocompatibility complex class IIB allele polymorphism and its association with resistance/susceptibility to Vibrio anguillarum in Japanese flounder (Paralichthys olivaceus)

The full length of major histocompatibility complex (MHC) class IIB cDNA was cloned from a Chinese population of Paralichthys olivaceus by homology cloning and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The MHC IIB genomic sequence is 1,864 bp long and consists of 34-bp 5′UTR, 741-bp open reading frame, 407-bp 3′UTR, 96-bp intron1, 392-bp intron2, 85-bp intron3, and 109-bp intron4. Phylogenetic analysis showed that the putative MHC class IIB amino acid of the Chinese P. olivaceus shared 28.3% to 85.4% identity with that of the reported MHC class IIB in other species. A significant association between MHC IIB polymorphism and disease resistance/susceptibility was found in Chinese P. olivaceus. Thirteen different MHC IIB alleles were identified among 411 clones from 84 individuals. Among the 280 (268) nucleotides, 32 (11.4%) nucleotide positions were variable. Most alleles such as alleles a, b, c, d, e, f, j, k, i, m were commonly found in both resistant and susceptible stock. Via χ² test, allele d was significantly more prevalent in individuals from susceptible stock than from resistant stock, and their percentages were 23.80% and 7.14%, respectively. In addition, allele g occurred in 9 and allele h in 4 of 42 resistant individuals that were not present in the susceptible stock; their percentages were 21.4% and 9.52%, respectively. Although allele l was found only in 8 individuals from the susceptible stock, its percentage is 19.05%.     

Molecular cloning,characterization and expression of a chalcone reductase gene from <Emphasis Type="Italic">Astragalus membranaceus</Emphasis> Bge. var. <Emphasis Type="Italic">mongholicus</Emphasis> (Bge.) Hsiao

A chalcone reductase (CHR) gene was isolated from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). The full-length cDNA of A. mongholicus CHR, designated as Amchr (GenBank accession No. HM357239), was 1196 bp long. It had a 957 bp open reading frame encoding a 318-amino acid protein of 35 kDa, a 67 bp 5′ non-coding region and a 172 bp 3′-untranslated region. The putative AmCHR protein showed striking similarity to CHR from other leguminous species. Two-dimensional structure modeling showed that AmCHR consisted of 45.28% α-helix, 10.38% extended strand and 44.34% random coil. Prediction showed that three-dimensional AmCHR was a global protein containing an aldo-ket-red domain, with a putative Asp-Tyr-Lys-His catalytic tetrad in the center. The AmCHR gene was 1251 bp long, consisting of three exons and two introns. Intron I was 125 bp and intron II was 169 bp long. Southern blot analysis indicated that Amchr belonged to a small multigene family. Under natural conditions, Amchr was expressed differentially in the root, stem and leaf tissues of A. mongholicus, with a preferential expression in the root. The recombinant AmCHR protein was successfully expressed in Escherichia coli strain BL21 with pET42a vector. The result showed that the expressed AmCHR protein had molecular weight of about 35 kDa, which matched the size of the predicted protein by bioinformatic analysis. This study opened avenues towards understanding of the function of AmCHR protein and the role of the Amchr gene in the calycosin-7-O-β-d-glucoside branch pathway in A. mongholicus.     

The First Complete cDNA Sequence of the Hemocyanin from a Bivalve,the Protobranch <Emphasis Type="Italic">Nucula nucleus</Emphasis>

By cDNA sequencing we have achieved the first, and complete, hemocyanin sequence of a bivalve (Nucula nucleus). This extracellular oxygen-binding protein consists of two immunologically distinguishable isoforms, here termed NnH1 and NnH2. They share a mean sequence identity of 61%, both contain a linear arrangement of eight paralogous, ca.50-kDa functional units (FUs a-h), and in both isoforms the C-terminal FU-h possesses an extension of ca. 100 amino acids. The cDNA of NnH1 comprises 11,090 bp, subdivided into a 5′utr of 75 bp, a 3′utr of 791 bp, and an open reading frame for a signal peptide of 19 amino acids plus a polypeptide of 3389 amino acids (M _r = 385 kDa). The cDNA of NnH2 comprises 10,849 bp, subdivided into a 5′utr of 47 bp, a 3′utr of 647 bp, and an open reading frame for a signal peptide of 16 amino acids plus a polypeptide of 3369 amino acids (M _r = 387 kDa). In contrast to other molluscan hemocyanins, which are highly glycosylated, the bivalve hemocyanin sequence exhibits only four potential N-glycosylation sites, and within both isoforms a peculiar indel is present, surrounding the highly conserved copper-binding site CuA. Phylogenetic analyses of NnH1 and NnH2, compared to the known hemocyanin sequences of gastropods and cephalopods, reveal a statistically sound closer relationship between gastropod and protobranch hemocyanin than to cephalopod hemocyanin. Assuming a molecular clock, the last common ancestor of protobranch and gastropods lived 494 million ± 50 million years ago, in conformity with fossil records from the late Cambrian. [Reviewing Editor: Dr. Rüdiger Cerff] The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ786639 for NnH1 and AJ786640 for NnH2.     

Identification and characterization of interferon regulatory factor-1 from orange-spotted grouper (<Emphasis Type="Italic">Epinephelus coioides</Emphasis>)

Interferon-regulatory factor 1 (IRF-1) is the first member of IRF family, which is involved in many biological processes such as immune response, antiviral defense, cell growth regulation, and apoptosis. In this study, an IRF-1 gene, EcIRF-1, was isolated and characterized from orange-spotted grouper (Epinephelus coioides). The full-length cDNA of EcIRF-1 is 1,730 bp, including an open reading frame of 906 bp, a 5′-terminal untranslated region (5′-UTR) of 153 bp, and a 3′-UTR of 671 bp. The EcIRF-1 gene consists of 10 exons and 9 introns, spanning over approximate 4.3 kb of genomic sequence. The 5′-UTR sequence contains an exon and an intron, and the 3′-UTR sequence is included in the last exon. Expression analysis by real-time PCR reveals that the EcIRF-1 gene is ubiquitously expressed in various healthy fish tissues, whereas its expression is upregulated in vivo in response to polyinosinic–polycytidylic acid or lipopolysaccharide stimulation. Subcellular localization analysis shows the EcIRF-1 is an intranuclearly localized and immobile protein in the cultured fish cells. Data presented in this paper provide an important base to further understand EcIRF-1 gene function and its regulation associated with interferon immune system in orange-spotted grouper.     

Molecular characterization,expression profile and association analysis with fat deposition traits of the porcine APOM gene

Apolipoprotein M (APOM), a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in plasma, is involved in lipid and lipoprotein metabolism. Through comparative mapping, we have mapped this gene to SSC7 p1.1 in which many QTLs affecting fat deposition traits have been reported. As a candidate gene for fat deposition traits, in this study, we obtained the 742-bp mRNA sequence of porcine APOM including the full coding region and encoding a protein of 188 amino acids. The sequence was deposited into the GenBank under the accession no. DQ329240. Semi-quantitative RT-PCR results showed that the porcine APOM gene is expressed predominantly in liver and kidney tissue. The genomic sequence of this gene which contains six exons and five introns, is 3,621 bp in length (DQ272488). Bioinformatic analysis of the 5′ regulatory region has revealed that classical TATA-box element and species conserved Hepatocyte nuclear factor-1a (HNF-1α) biding site were represented in this region. A G2289C single nucleotide polymorphism (SNP) in the intron 2 of porcine APOM gene detected as an Eco130I PCR–restriction fragment length polymorphism (PCR–RFLP) showed allele frequency differences among three purebreds. Association of the genotypes with fat deposition traits showed that different genotypes of porcine APOM gene were significantly associated with leaf fat weight (P < 0.05), backfat thickness at shoulder (P < 0.05), backfat thickness at thorax-waist (P < 0.05), backfat thickness at buttock (P < 0.01) and average backfat thickness over shoulder, thorax-waist and buttock (P < 0.01).     

Molecular cloning and characterization of the <Emphasis Type="Italic">Dicer-like</Emphasis> 2 gene from <Emphasis Type="Italic">Brassica rapa</Emphasis>

Dicer-like proteins (DCLs) are involved in small RNA-mediated development and viral defense in plants. In model plants, at least four DCLs have been found and a number of studies have helped to understand their function. However, the function of the Dicer or DCLs in other plants is still unclear. Here, we report the full-length cDNA sequence of Brassica rapa ssp. chinensis DCL2 (BrDCL2) gene, which contains a 4,179 bp open reading frame (ORF) encoding a protein of 1,392 amino acids. At the 3′ end of BrDCL2, clones with three different lengths of 3′ untranslated region were found. An alternative splice variant of BrDCL2, BrDCL2sv, in which one intron was retained between exon9 and exon10, was also cloned. Because of a change in the coding sequence resulting in a premature terminal codon, BrDCL2sv was expected to translate a short peptide containing the whole DEXHc domain.     

Colonization routes of Pinus sylvestris inferred from distribution of mitochondrial DNA variation

Understanding the present-day distribution of molecular variation requires knowledge about the history of the species. Past colonization routes and locations of refugia of Scots pine (Pinus sylvestris) were inferred from variation in mitochondrial DNA in material collected from 37 populations located in countries within, and immediately adjacent to the continent of Europe. Two mitochondrial regions, nad1 intron (exon B/C) and nad7 intron 1, were included in the study. Differentiation in maternally inherited mitochondria was high (G _ST′ = 0.824). Two new haplotypes were found at the nad7 intron 1. The occurrence of a 5-bp indel variant was restricted to the Turkish Kalabak population and a 32 bp only found in Central, Eastern, and Northern Europe. The complete absence of the 32-bp indel from the Mediterranean peninsulas supports the view that coniferous forests existed outside these areas during the last glacial maximum, and these populations contributed to the subsequent colonization of the northern parts of Europe. P. sylvestris shares features of its glacial and postglacial history with two other northern, cold-tolerant tree species, Picea abies and Betula sp. These three species differ from many other European trees for which pollen core and molecular evidence indicate colonization from southern refugia after the last glacial period.     

Characterization of the tissue-specific expression of phenylalanine ammonia-lyase gene promoter from loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

We isolated the 5′ flanking region of a gene for phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Pinus taeda, PtaPAL. To investigate the tissue-specific expression of the PtaPAL promoter, histochemical assay of GUS activity was performed using the transgenic tobacco expressing the PtaPAL promoter-GUS. The region of −897 to −420 in PtaPAL promoter showed high activities in the secondary xylem and response to bending stress. To characterize the cis-regulatory functions of the promoters for enzymes in phenylpropanoid biosynthesis, we examined the activity of chimeric promoters of PtaPAL and a 4-coumarate CoA ligase, Pta4CLα. The chimeric promoter showed similar activity as the Pta4CLα promoter. Electrophoretic mobility shift assays implicated −897 to –674 of PtaPAL promoter containing cis-elements of the expression in xylem of Pinus taeda. The results suggested that AC elements of PtaPAL have multiple functions in the expression under the various developmental stages and stress conditions in the transgenic tobacco. The nucleotide sequence data reported will appear in the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under the accession number AB449103 (PtaPAL promoter sequence).     

Molecular Basis of the Size Polymorphism of the First Intron of the<Emphasis Type="Italic">Adh-1</Emphasis> Gene of the Mediterranean Fruit Fly, <Emphasis Type="Italic">Ceratitis capitata</Emphasis>

The first intron of the gene encoding one of the alcohol dehydrogenase isoenzymes (ADH-1) in Ceratitis capitata is highly polymorphic in size. Five size variants of this intron were isolated from different strains and populations and characterized. Restriction map and sequence analysis showed that the intron size polymorphism is due to the presence or absence of (a) a copy of a defective mariner-like element, postdoc; (b) an 550-bp 3 indel which exhibits no similarity to any known sequence; and (c) a central duplication of 704 bp consisting of part of the 3 end of the postdoc element, the region between postdoc and the 3 indel, and the first 20 bp of the 3 indel. The homologous Adh-1 intron was amplified from the congeneric species, Ceratitis rosa, in order to obtain an outgroup for comparative and phylogenetic analyses. The C. rosa introns were polymorphic in size, ranging from about 1100 to 2000 bp, the major difference between them being the presence or absence of a mariner-like element Crmar2, unrelated to the postdoc element. Phylogenetic analysis suggests that the shorter intron variants in C. capitata may represent the ancestral form of the intron, the longest variants apparently being the most recent.