Molecular cloning and characterization of a 2C-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-erythritol 2,4-cyclodiphosphate synthase gene from <Emphasis Type="Italic">Cephalotaxus harringtonia</Emphasis>

The full-length MECPS cDNA sequence (designated as Chmecps, GenBank Accession No.: DQ415658) was isolated by rapid amplification of cDNA ends (RACE) for the first time from Cephalotaxus harringtonia. The full-length cDNA of Chmecps was 1,146 bp containing a 753 bp open reading frame (ORF) encoding a polypeptide of 250 amino acids with a calculated mass of 26.67 kDa and an isoelectric point of 9.35. Comparative and bioinformatics analyses revealed that ChMECPS showed extensive homology with MECPSs from other plant species. Phylogenetic analysis indicated ChMECPS was more ancient than other plant MECPSs. Southern hybridization analysis of the genomic DNA showed that Chmecps was a single copy gene. Tissue expression pattern analysis revealed that ChMECPS expressed strongly in root and leaf, weakly in stem.     

Molecular cloning,characterization and expression analysis of <Emphasis Type="Italic">Cm</Emphasis>APX

The RT PCR and RACE methods were used to obtain the cDNA sequence of an APX gene of muskmelon after the leaves were induced with powdery mildew. The cDNA length of the APX gene is 1,047 bp with a 750 bp ORF encoded a 249 amino acid and the molecular weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns. The identity of the amino acid sequence deduced from the cDNA with the APX family of other homologous members was about 74–97%. A Full-length of ORF was sub-cloned into prokaryotic expression vector pET24a. The recombinant proteins had high expression level in E. coli. Analysis of expression at mRNA level showed that CmAPX exhibited highly tissue-specific patterns of expression. The mRNA level and enzyme activities assays showed that CmAPX might play an important role in the pathogenesis of powdery mildew.     

Cloning and expression of arginine kinase from a swimming crab, <Emphasis Type="Italic">Portunus trituberculatus</Emphasis>

Arginine kinase (AK) is an important phosphotransferase that plays a critical role in energy metabolism in invertebrates. In this paper, the cDNA of AK (designated as PtAK) was identified from the eyestalk cDNA library of swimming crab Portunus trituberculatus. The full-length cDNA was 1,479 bp, containing an open reading frame of 1,074 bp that coded for 357 amino acids. The estimated molecular mass of mature PtAK was 40.30 kDa and theoretical isoelectric point was 6.18. Amino acid sequence alignment showed that PtAK had very high similarity with other shrimp and crab AKs ranging from 0.876 to 0.983. The genomic DNA fragments of about 1,434 bp consisted of two exons interrupted by an intron. Totally 24 SNPs, including 17 in the coding region and seven in the non-coding region, were detected by direct sequencing of 19 genomic samples. In exon 1, the coding SNPs (cSNPs) were only found in the disease-resistant specimens. The fluorescent real-time PCR analysis revealed that the expression of PtAK was detected in all the examined tissues with the highest expression in the muscle and the lowest in the eyestalk. The expression of PtAK after Vibrio alginolyticus injection was tested in haemocytes, showing that two peak values were 5.01-fold (at 3 h) and 3.60-fold (at 24 h) compared with the control values, respectively. The results suggested that AK might play an important role in the immune response in crabs.     

Molecular cloning and expression analysis of a new putative gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

A new putative gene encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase (designated as SmHMGS, GenBank Accession No. FJ785326), which catalyses the condensation of acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the mevalonic acid pathway, was isolated from young leaves of Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of the putative SmHMGS was 1,655 bp containing a 1,381 bp open reading frame (ORF) encoding a polypeptide of 460 amino acids. Comparative and bioinformatic analyses revealed that SmHMGS showed extensive homology with HMGSs from other plant species. Phylogenetic tree analysis indicated that SmHMGS belonged to the plant HMGS super family and had the closest relationship with HMGS from Hevea brasiliensis. Tissue expression pattern analysis revealed that the putative SmHMGS was constitutively expressed in all the tested tissues and strong in leaf, moderate in stem, weak in root, which was in contrast to SmHMGR reported before. The putative SmHMGS was found to be an elicitor-responsive gene, which could be induced by exogenous elicitors, including salicylic acid (SA) and methyl jasmonate (MJ). These results will help in understanding the role of HMGS in tanshinones biosynthesis in S. miltiorrhiza.     

Molecular cloning,characterization and expression analysis of a new gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is the first committed step in MVA pathway for isoprenoid biosynthesis in plants. In this study, a full-length cDNA encoding HMGR was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmHMGR (GenBank Accession No.EU680958). The full-length cDNA of SmHMGR was 2,115 bp containing a 1,695 bp open reading frame (ORF) encoding a polypeptide of 565 amino acids. Bioinformatic analyzes revealed that the deduced SmHMGR had extensive homology with other plant HMGRs contained two transmembrane domains and a catalytic domain. Molecular modeling showed that SmHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. Phylogenetic tree analysis indicated that SmHMGR belongs to the plant HMGR super-family and has the closest relationship with HMGR from Picrorhiza kurrooa. Expression pattern analysis implied that SmHMGR expressed highest in root, followed by stem and leaf. The expression of SmHMGR could be up-regulated by salicylic acid (SA) and methyl jasmonate (MeJA), suggesting that SmHMGR was elicitor-responsive. This work will be helpful to understand more about the role of HMGR involved in the tanshinones biosynthesis at the molecular level.     

Molecular cloning and expression analysis of an F-box protein gene responsive to plant hormones in <Emphasis Type="Italic">Brassica napus</Emphasis>

F-box protein family is characterized by an F-box motif that has been shown to be critical for the controlled degradation of regulatory proteins. In plant, F-box protein plays an important role in signal pathways and involved in various signal transduction systems. A full-length cDNA encoding a putative F-box protein, designated as BnSLY1, was isolated from Brassica napus. The full-length cDNA of BnSLY1 was 809 bp containing a 438 bp open reading frame encoding a precursor protein of 138 amino acid residues. Comparative and bioinformatic analyses revealed that BnSLY1 showed high degree of homology with F-box proteins from other plant species and contained F-box, GGF and LSL conserved motifs. The expression of BnSLY1 under exogenous gibberellins acid-3 (GA₃), abscisic acid (ABA) and GA biosynthetic inhibitor paclobutrazol (PAC) was analyzed using real-time PCR. The results showed that the expression of BnSLY1 was down-regulated after GA₃ treatment and prominently induced by ABA in the low concentrations. Moreover, BnSLY1 was also induction in the high concentrations of PAC. These results suggest that the expression of BnSLY1 was regulated by the exogenous GA₃, ABA and PAC and may be related to endogenous level of GA in B. napus.     

Cloning,characterization and expression of two new polyphenol oxidase cDNAs from <Emphasis Type="Italic">Agaricus bisporus</Emphasis>

Two new polyphenol oxidase (PPO) cDNAs (PPO3 and PPO4 cDNAs, accession numbers GQ354801 and GQ354802, respectively) were obtained by RACE-PCR from Agaricus bisporus. PPO3 cDNA was 1844 bp in length with an open reading frame of 1731 bp, while PPO4 cDNA was 2042 bp with an open reading frame of 1836 bp. PPO3 and PPO4 cDNAs, with 52% identity at the nucleic acid level, encoded a 576-amino acid protein of 66.3 kDa and 611-amino acid protein of 68.3 kDa, respectively. Mature forms of PPO3 and PPO4 were characterized after removing the specific C-terminal region and expressed in Escherichia coli BL21 (DE3) RIPL using pGEX-4T-1 vector. The expressed proteins were probed by the anti-A. bisporus PPO antibody but without PPO activity. This indicated that the recombinant mature PPO3 and mature PPO4 could not form an active center in prokaryotic expression system.     

Cloning and sequence analysis of an acidophilic xylanase (XynI) gene from <Emphasis Type="Italic">Aspergillus usamii</Emphasis> E001

The full-length cDNA gene that encodes an acidophilic endo-1,4-β- xylanase XynI of Aspergillus usamii E001 was amplified by rapid amplification of cDNA 3′ and 5′ ends (RACE) using the total RNA as template and then cloned onto the pUCm-T vector, followed by sequencing. The cloned cDNA is 881 bp in length including 5′ and 3′ non-encoding regions, as well as a 678 bp of open reading frame (ORF) which encodes an E001 XynI of 188 amino acid residues together with a signal peptide of 37 amino acid residues. The homologies of E001 XynI with xylanases of Aspergillus niger, Aspergillus kawachii, Emericella nidulans and Penicillium funiculosum are 97.8, 92.0, 74.6 and 60.5%, respectively. From a BLAST search result, we concluded that E001 XynI belongs to the glycoside hydrolase family 11. Its three-dimensional structure was predicted using programs based on that of the P. funiculosum xylanase (1TE1B) from the family 11. In addition, the complete DNA gene xynI encoding E001 XynI was cloned from the genomic DNA of A. usamii E001 by conventional PCR and ligation-mediated PCR amplification. The cloned xynI is 1,206 bp in length, composed of a promoter region, a 68 bp of intron and two exons when compared with the cDNA of E001 XynI.     

Molecular analysis of a ras-like nuclear (Ran) gene from <Emphasis Type="Italic">Penaeus monodon</Emphasis> and its expression at the different ovarian stages of development

In the present study, a ras-like nuclear (Ran) gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of black tiger prawn (Penaeus monodon). The full-length black tiger prawn Ran (PmRan) cDNA consisted of 1140 nucleotides including an open reading frame (ORF) 648 bp, a 5′ untranslated region (5′UTR) of 117 bp and a 3′UTR of 375 bp with a polyadenylation signal sequence “aataaa” and a poly (A) tail. The ORF encoded a peptide of 215 amino acids with molecular mass 24.6 kDa and a theoretical isoelectric point of 7.39. ScanProsite analysis indicated that PmRan protein sequence contained a small GTPase Ran family motif. Homology analysis of the deduced amino acid sequence of the PmRan with other known Ran sequences by MatGAT software revealed that the PmRan show very high homology with the sequences of other animals (92.1–98.6% similarity, 85.6–98.1% identity). Analysis of the tissue expression pattern of the PmRan gene showed that the PmRan mRNA was expressed in all tested tissues, including hepatopancreas, ovary, muscle, intestine, neurosecretory organ in eyestalk, neurosecretory organ in brain, stomach, and heart, with the highest levels in ovary. Furthermore, the PmRan expression was found to be high level in the six ovarian stages of development. The results indicated PmRan might play an important role in ovarian development.     

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.     

AGAMOUS‐LIKE13, a putative ancestor for the E functional genes,specifies male and female gametophyte morphogenesis

Arabidopsis AGL13 is a member of the AGL6 clade of the MADS box gene family. GUS activity was specifically detected from the initiation to maturation of both pollen and ovules in AGL13:GUS Arabidopsis. The sterility of the flower with defective pollen and ovules was found in AGL13 RNAi knockdown and AGL13 + SRDX dominant‐negative mutants. These results indicate that AGL13 acts as an activator in regulation of early initiation and further development of pollen and ovules. The production of similar floral organ defects in the severe AGL13 + SRDX and SEP2 + SRDX plants and the similar enhancement of AG nuclear localization efficiency by AGL13 and SEP3 proteins suggest a similar function for AGL13 and E functional SEP proteins. Additional fluorescence resonance energy transfer (FRET) analysis indicated that, similar to SEP proteins, AGL13 is able to interact with AG to form quartet‐like complexes (AGL13–AG)₂ and interact with AG–AP3–PI to form a higher‐order heterotetrameric complex (AGL13–AG–AP3–PI). Through these complexes, AGL13 and AG could regulate the expression of similar downstream genes involved in pollen morphogenesis, anther cell layer formation and the ovule development. AGL13 also regulates AG/AP3/PI expression by positive regulatory feedback loops and suppresses its own expression through negative regulatory feedback loops by activating AGL6, which acts as a repressor of AGL13. Our data suggest that AGL13 is likely a putative ancestor for the E functional genes which specifies male and female gametophyte morphogenesis in plants during evolution.     

Cloning,expression and characterization of the putative nuclear transport factor 2 (NTF2) gene from moss <Emphasis Type="Italic">Conocephalum conicum</Emphasis>(L.) Dum

Biomacromolecules import into the nucleus is a complex progress which requires the participation of several cytosolic factors, and nuclear transport factor 2 (NTF2) is one of essential components in nuclear trafficking. Its main role is to transport RanGDP from cytoplasm to nucleus by interacting with FxFG nucleoporin repeats. In the study a putative new gene, designated as CcNTF2, was obtained from the moss (Conocephalum conicum) cDNA library we have constructed. The full-length cDNA sequence is 913 bp in size contains a 372 bp open reading frame (ORF) flanked by a 195 bp 5′-untranslated sequence and a long 346 bp 3′-non-coding region, encoding 123 amino acids of 13,575.3 Da. Part of the genomic sequence was also cloned and sequenced, which is 1,602 bp long and possesses two exons and one intron. Alignment analysis showed that the CcNTF2 protein is high conserved among plant NTF2 and shares 81% similarity with the ones from Arabidopsis thaliana and Brassica rapa. The expression of wild-type CcNTF2 was detected by immunoblotting of extraction of C. conicum and it indicated the putative protein is integral. Through functional expression of CcNTF2-green fluorescent protein (GFP) in tobacco, it was demonstrated that CcNTF2 can accumulate at the nuclear rim. Site-directed mutagenesis analysis confirmed CcNTF2 P71K has influence on the protein import into nucleus. In addition, overexpression of CcNTF2 P71K was observed to be deleterious for the plant cell. It is the first illumination of NTF2 in moss, and our study established the primary foundation for further research on moss NTF2.     

Molecular characterization and expression analysis of a new cDNA encoding strictosidine synthase from <Emphasis Type="Italic">Ophiorrhiza japonica</Emphasis>

A new full-length cDNA encoding strictosidine synthase (designated as OjSTR, GenBank Accession No. 1087598), which catalyzes a committed step in camptothecin biosynthetic pathway, was isolated from young leaves of Ophiorrhiza japonica for the first time. OjSTR was 1,258 bp and contained a 1,062 bp open reading frame encoding a deduced protein of 353 amino acid residues. Sequence analyses showed that OjSTR had high homology with other STRs from some TIA-producing plants. Phylogenetic tree analysis showed that OjSTR had closest relationship with STR from O. pumila. Tissue expression pattern analysis revealed that OjSTR constitutively expressed in all the tested tissues at different levels, which was high in flower, moderate in leaf and root, low in stem. Expression profiles under plant defense signals such as methyl jasmonate and salicylic acid were investigated, and the results revealed that expression of OjSTR was all induced, implying that OjSTR was high elicitor responsive.     

Partial molecular characterization,expression pattern,polymorphism and association analysis of porcine <Emphasis Type="Italic">SKP2</Emphasis> gene

As a component of E3 ubiquitin protein ligases called SCFs, SKP2 protein belongs to a member of FBLs protein which is the biggest eukaryotic subfamily of F-BOX proteins with 12 members. In this study, we cloned and sequenced partial cDNA, intron 1 and intron 6 of porcine SKP2 gene. The partial cDNA is 1,402 bp long and has an open reading frame of 1,272 bp which encodes 424 putative amino acids. The deduced protein comprises a conserved F-BOX domain at position from the 90th to 140th amino acid. The phylogenetic tree indicated that porcine SKP2 has the closest genetic relationship with bovine SKP2 than other selected animal species. Quantitative RT-PCR analysis displayed that the tissue expression level of porcine SKP2 fluctuated remarkably in a large range, and it expressed in thymus with the highest level and in longissimus dorsi muscle with the lowest level. Two SNPs were identified, meanwhile, further polymorphism analysis with Cfr42I showed that AA genotype was in dominance absolutely among four kinds of unrelated Chinese indigenous miniature and one introduced Landrace pig breeds. In addition, association analysis with immune traits and blood parameters revealed that the SNP Cfr42I in intron 1 was significantly associated with red cell distribution width of neonate piglets at 0 day (P = 0.027).