Molecular cloning, sequence identification, and expression analysis of HMGR gene in Houttuynia cordata / 中草药

Objective To clone and analyze the expression difference sequence of 3-hydroxy-3-methylglutaryl coenzyme A redutase (HMGR) gene from Houttuynia cordata. Methods The sequence of HMGR was cloned from H. cordata by RT-PCR strategy. The physical and chemical properties, secondary structure, and three-dimensional structure of the HMGR protein were forecasted and analyzed, and its structure and function were predicted. And the different expression of HMGR gene in rhizome, stems, leaves, and flowers were analyzed by fluorescent quantitative PCR. Results The cDNA contained a 1 626 bp open reading frame and encoded a predicted protein of 541 amino acids. Two transmembrane regions and no signal peptide were present in HMGR. Relative real-time PCR analysis indicated that HMGR showed the highest transcript abundance in the flowers, and the lowest levels in the rhizomes. Conclusion This study cloned and expression analyzed HMGR gene from H. cordata for the first time. The result will provide a foundation for exploring the mechanism of terpenoid biosynthesis in H. cordata.

Cloning, expression, and bioinformatics analysis of acetyl-CoA C-acetyltransferase gene in Houttuynia cordata / 中草药

Objective: Acetyl-CoA C-acetyltransferase (AACT) is the initial enzyme in the terpenoid biosynthesis pathway of mevalonate (MVA), two units of acetyl-CoA were catalyzed to acetoacetyl-CoA. To clone the full length cDNA of AACT gene and carry out the bioinformatics analysis and expression analysis in order to provide the basis on resolving the mechanism of biosynthesis for terpenoid secondary metabolites from Houttuynia cordata. Methods: The cDNA sequence of AACT gene was obtained from H. cordata by using RT-PCR strategy. And the different expression of AACT gene in the rhizomes, stems, leaves, and flowers of H. cordata was analyzed by fluorescent quantitative PCR. Results: The cDNA contains a 1 218 bp open reading frame and encodes a predicted protein of 405 amino acids. No transmembrane region and signal peptide were present in AACT protein by bioinformatics prediction. Relative real-time PCR analysis indicated that AACT gene showed the highest transcript abundance in the stems and rhizomes of H. cordata lower levels in the flowers and leaves, the values of them were 1.49, 0.96, 0.20, and 0.10, respectively. Conclusion: This AACT gene is cloned from H. cordata for the first time. The results will provide a foundation for exploring the mechanism of the gene in terpenoid biosynthesis and metabolism in H. cordata.

Molecular cloning, differential expression, and bioinformatics analysis of HMGR gene in Sambucus chinensis / 中草药

Objective: To clone the 3-hydroxy-3-methylglutaryl coenzyme A redutase (HMGR) gene from Sambucus chinensis and analyze the differential expression. Methods: The sequence of HMGR was cloned from S. chinensis by using RT-PCR strategy. The physical and chemical properties, secondary structure, and tertiary structure of the HMGR protein were forecasted and analyzed, and its structure and function were predicted. And the different expression of HMGR gene in the rhizome, stems, leaves, and flowers was analyzed by fluorescent quantitative PCR. Results: The cDNA contains a 1782 bp open reading frame and encodes a predicted protein of 593 amino acids. Two transmembrane regions and no signal peptide were present in HMGR. Relative real-time PCR analysis indicated that HMGR showed the higher transcript abundance in the flowers and aerial stems, and the lower levels in the rhizomes and leaves. Conclusion: This study clones and expression analyzes HMGR gene from S. chinensis for the first time. The results will provide a foundation for exploring the mechanism of terpenoid biosynthesis in S. chinensis.

Cloning and differential expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene in Houttuynia cordata / 中草药

Objective: To clone the 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene from Houttuynia cordata and analyze the differential expression. Methods: The cDNA sequence of DXR was cloned from H. cordata by using RT-PCR strategy. The physical and chemical properties, secondary structure, and three-dimensional structure of the DXR protein were forecasted and analyzed, and its function was predicted. The differential expression of DXR gene in rhizome, stems, leaves, and flowers was analyzed by fluorescent quantitative PCR. Results: The cDNA contained a 1 416 bp open reading frame and encoded a predicted protein of 471 amino acids. Bioinformatics predicted that no transmembrane region and signal peptide were present in DXR. Relative real-time PCR analysis indicated that DXR showed the highest transcript abundance in leaves, moderate level in rhizomes, lower level in stems, and the lowest level in flowers. Conclusion: This study clones DXR gene from H. cordata for the first time, and provides a foundation for exploring the mechanism of this gene for the terpenoid biosynthesis in H. cordata.

Gene cloning of anthocyanin synthase in Fagopyrum dibotrys and correlation between its expression level and anthocyanin content / 中草药

Objective: To obtain the full-length cDNA sequence of anthocyanins synthase (ANS) gene from Fagopyrum dibotrys (FdANS), and to analyze the expression of FdANS in each tissue and the total anthocyanin content during florescence of F. dibotrys. Methods: The cDNA sequence of ANS gene was cloned by homology cloning from F. dibotrys. The expression of ANS was analyzed by semi-quantitative RT-PCR. The content of total anthocyanin was measured by spectrophotometry. Results: The open reading frame (ORF) of FdANS was 1077 bp and encoded a protein with 358 amino acids named FdANS. Bioinforamtion analysis suggested that the amino acid sequence of FdANS had the higher homology with those in other plants. Both the gene expression FdANS in different tissues during florescence of F. dibotrys and the total anthocyanin content were in the order of flowers > leaves > stems> roots. Conclusion: The cDNA sequence of FdANS is firstly obtained from F. dibotrys and its coding protein has the typical characteristic of ANS homologous protein. The gene expression of FdANS shows the correlation to the total anthocyanins content in the flowers, leaves, stems, and roots of F. dibotrys.

Cloning and expression analysis of 1-deoxy-D-xylulose-5-phosphate synthase 1 gene in Houttuynia cordata / 中草药

Objective: To clone the 1-deoxy-D-xylulose-5-phosphate synthase 1 (DXS1) gene from Houttuynia cordata and to analyze the expression difference. Methods: The cloning primers were designed based on the transcriptome dataset of H. cordata, one unique sequence encoding DXS1 was discovered. The sequence of DXS1 was cloned from H. cordata by RT-PCR. The physicochemical properties, secondary structure, and three-dimensional structure of the DXS1 protein were forecasted and analyzed, and its structure and function were predicted. And the different expression levels of DXS1 gene in rhizome, stems, leaves, and flowers were analyzed by fluorescent quantitative PCR. Results: The cDNA (named as DXS1) contains a 2 172 bp open reading frame and encodes a predicted protein of 723 amino acids. No transmembrane region and signal peptide were present in DXS1. The conserved domain of DXS was present in DXS1. Relative real-time PCR analysis indicated that DXS1 showed the highest transcript abundance in the flowers, moderate level in the leaves, lower level in the rhizomes, and the lowest level in the stems. Conclusion: This study cloned the DXS1 gene from H. cordata for the first time. The results will lay a foundation for exploring the mechanism of terpenoid biosynthesis in H. cordata plants.

Acquisition and Analysis of Three cDNA Full-Length Sequences from the Liver of Microtus fortis / 中国实验动物学报

Objective To obtain the full-length cDNA sequences of CYP2E1,CYP2D5,ECHS1,which may be related with non-alcoholic fatty liver disease,from Microtus fortis.Methods To construct Microtus fortis liver cDNA plasmid library using SMART technique,to get the purposed colonies through screening libraries by PCR,and to obtain their full-length cDNA sequences by sequencing with pBluescript II SK universal primers M13R.Results Three full-length cDNA sequences of Microtus fortis,CYP2E1,CYP2D5 and ECHS1 were obtained.The CYP2E1 cDNA was 1685 bp in length and contained a 1482 bp open reading frame(ORF) encoding a 494 amino acids.The CYP2D5 cDNA was 1690 bp in length,and contained a 1514 bp ORF encoding 504 amino acids.The ECHS1 cDNA was 1013 bp in length,and containsed an 873 bp ORF encoding 290 amino acids.Sequence analysis revealed that the identity of the three cDNA sequences and deduced amino acids among Microtus fortis,Homo sapiens,Mus musculus and Rattus norvegicus was high.Conclusion The full-length cDNA sequences of CYP2E1,CYP2D5,ECHS1 were obtained from Microtus forti,liver cDNA library.and the gene sequences have been deposited in GenBank (GQ507485,GQ507486,GQ845171),which may lay the foundation for researchies of pathogenesis of non-alcoholic fatty liver disease in Microtus fortis models.

Interaction between Parasitophorous Vacuolar Membraneassociated GRA3 and Calcium Modulating Ligand of Host Cell Endoplasmic Reticulum in the Parasitism of Toxoplasma gondii

A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.

Molecular cloning of a rhoptry protein (ROP6) secreted from Toxoplasma gondii

Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3'-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5'-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.

Molecular cloning of ribosomal P protein in Toxoplasma gondii and the availability to detect antibody against recombinant protein in toxoplasmosis patients

Among the panel of monoclonal antibodies (mAb) against Toxoplasma gondii, mAb of Tg621 (Tg621) clone blotted 38 kDa protein which localized in the cytoplasm of tachyzoites by immunofluorescence microscopy. The protein was not released into the parasitophorous vacuole during or after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg621. The full length cDNA sequence was completed with 5'-RACE as 1, 592 bp, which contained open reading frame of 942 bp. The deduced amino acid sequence of Tg621 consisted of a polypeptide of 313 amino acids, with significant homology to ribosomal P proteins (RPP) of other organisms especially high to those of apicomplexan species. The expressed and purified TgRPP was assayed in western blot with the sera of toxoplasmosis patients and normal sera, which resulted in the 74.0% of positive reactions in toxoplasmosis patients whereas 8.3% in normal group. Therefore, the antibody formation against TgRPP in toxoplasmosis patients was regarded as specific for T. gondii infection and suggested a potential autoantibody.

Sequence analysis of full length cDNA of Schistosoma japonicum egg miracidia genes harboring signal sequence / 中国血吸虫病防治杂志

Objective To analyze the full length cDNA sequence of Schistosoma japonicum egg miracidia genes harboring signal sequence.Methods The gene specific primers were designed and synthesized according to S.japonicum egg miracidia cDNA fragment containing signal sequence identified by signal sequence trapping method previously. The 5′and 3′ end cDNA fragments of each egg miracidia cDNA fragment harboring signal sequence were amplified by nest PCR using the first strand cDNA of S.japonicum as the template. The specific PCR fragments were cloned by TA clone method and sequenced. The full length cDNA sequence of each gene with signal sequence was constructed by comparing the cDNA sequence identified with signal sequence trapping method and the 5′ end sequence, the 3′ end sequence and deleting the overlapping fragments. The splicing model between mRNA of signal sequence and one of mature portions of S.japonicum egg miracidia gene was checked by analyzing the genomic DNA sequence structure of some genes with signal sequence. Results The 5′ and 3′ end cDNA fragments of sixteen among thirty cDNA fragment with signal sequence were amplified successfully, and their DNA sequences were determined. The full length cDNA sequences of sixteen egg miracidia genes were obtained by sequence matching and splicing. The results of deduced amino acid analysis found that the signal peptide of gene SjP4001 was the same to the one of SjP1531 and the signal peptide of gene SjP1183 was similar to the one of gene SjP3742. It confirmed that different genes could share the same or similar signal peptide. The data of S.japonicum genomic DNA sequence analysis showed that the S.japonicum could obtain its signal sequence by alternative splicing model or trans-splicing model. Conclusions The full length cDNA sequences of sixteen S.japonicum egg miracidia genes with signal sequence have been defined, it indicated that the S.japonicum egg miracidia genes could get its signal sequence by alternative splicing model or trans-splicing model was found in this study.

Cloning and Expression of the Gene Encoding Schistosoma japonicum Tropomyosin * / 中国寄生虫学与寄生虫病杂志

Objective To clone and express the cDNA encoding Schistosoma japonicum tropomyosin. Methods The cDNA was amplified by reverse transcription polymerase chain reaction (RT PCR). The PCR products were ligated with pGEM T vectors and then for transformations. After characterization of white clones by agarose gel electrophoresis, endonucleases digestion and PCR, some recombinant plasmids with inserts were used for sequencing. Then the gene was subcloned into prokaryotic expression vector pQE30 and expression was induced by IPTG. Results The PCR products was 823 bp judged by agarose gel electrophoresis and sequencing. A cDNA encoding S japonicum tropomyosin, except for 14 amino acids at the amino terminus and 2 at the carboxyl terminus, has been constructed and cloned successfully. The colony, designated pGSjcTM12, was sequenced and shown to be 91 1% identical at the nuclei acid level and 98.1% identical in deduced amino acid sequence to that of S mansoni tropomyosin. The gene was subcloned into pQE30 and an expressed protein of about 32 kDa was obtained.Conclusion The cloning and expression of the gene encoding S japonicum tropomyosin had been successfully made.