No abstract available.
Cloning, Molecular* ; Humans* ; Oxidoreductases*

Aims: Expression of recombinant proteins across a range of different host organisms has profound contribution to the advancement in biotechnology. In this study, we aimed to construct a highly versatile broad host range (BHR) expression vector, designated as pYL101C. Methodology and results: The Golden Gate cloning approach was used to construct pYL101C. Key features of pYL101C include a strong integron promoter (PINTc), a BHR pBBR1 origin of replication (ori), gentamycin resistance gene (GmR) as a selectable marker and a multiple cloning site (MCS) downstream of the promoter for easy-cloning purpose. To verify the functionality of pYL101C, we cloned the superfolder green fluorescent protein (sfGFP) reporter gene into pYL101C and transferred the resultant recombinant plasmid pYL101C::sfGFP into various Gram-negative bacteria. Transformants obtained stably expressed strong green fluorescence under blue light excitation even without selection after four passages. Conclusion, significance and impact of study The constructed BHR expression vector, pYL101C and recombinant pYL101C::sfGFP are stable and can be used to monitor the presence of Gram-negative bacteria, such as endophytes and pathogens in their hosts and environment.
Host Specificity ; Plasmids ; Cloning, Molecular

No abstract available.
Animals ; Cloning, Molecular* ; Interleukin-2* ; Mice*

Oxidosqualene cyclases(OSCs), belonging to a multigene family, can convert a common precursor 2,3-oxidosqualene into various types of triterpene skeletons. In this study, primers were designed according to the analysis of Siraitia grosvenorii transcriptome data, and two OSC genes SgAS1(GenBank No. QDO67189.1) and SgAS2(GenBank No. QDO67190.1) were cloned. The open reading frame(ORF) of SgAS1 was 2 262 bp, encoding 754 amino acids, and the ORF of SgAS2 was 2 289 bp, encoding 762 amino acids. Real-time quantitative PCR results demonstrated that the two SgOSCs genes showed different expression patterns in stems, leaves, and different stages of fruits. Phylogenetic analysis showed that both SgAS1 and SgAS2 were clustered with β-amyrin synthases into a branch, but further functional characterization using yeast heterologous expression found that SgAS1 was inactive and SgAS2 could produce β-amyrin as the sole product. Multiple sequence alignments revealed that SgAS2 had a conserved MWCYCR sequence related to β-amyrin biosynthesis, while SgAS1 had an unusual LFCYTR sequence, for which the authors performed site-directed mutagenesis analysis of this sequence and found that tryptophan residue(W) was the key amino acid residue that affected the function of SgOSCs. In addition, the authors transformed the monofunctional β-amyrin synthase SgAS2 into the chassis strain GH1, which was previously modified by the research group, and increased the yield of β-amyrin to 44.05 mg·L~(-1). This study first reported the monofunctional β-amyrin synthase SgAS2 from S. grosvenorii and conducted site-directed mutagenesis and synthetic biology investigation on it, providing a valuable resource for the directed biosynthesis of triterpenoids.
Phylogeny ; Triterpenes/metabolism* ; Cloning, Molecular ; Amino Acids

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.
Gastrodia/genetics* ; Phylogeny ; Amino Acids ; Cloning, Molecular

The modern bio-fermentation industry requires design and creation of efficient microbial cell factories for directed conversion of raw materials to target products. The main criteria for assessing the performance of microbial cell factories are their product synthesis capacity and stability. Due to the deficiencies of plasmids in gene expression such as instability and being easy to lose, integration of genes into chromosome is often a better choice for stable expression in microbial hosts. To this end, chromosomal gene integration technology has received much attention and has developed rapidly. In this review, we summarize the recent research progresses of chromosomal integration of large DNA fragments in microorganisms, illustrate the principles and features of various technologies, highlight the opportunity brought by the CRISPR-associated transposon systems, and prospect future research direction of this technology.
Chromosomes ; Plasmids ; DNA ; Cloning, Molecular ; Fermentation

OBJECTIVETo study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF).

METHODSHDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST.

RESULTSCloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms.

CONCLUSIONThe biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

No abstract available.
Cloning, Molecular* ; DNA* ; Helicobacter pylori* ; Helicobacter* ; Sequence Analysis, DNA* ; Urease*

The development of the genetic transformation systems in extremely halophilic Archaea was reviewed in this paper. Included are the screening of selectable markers for resistance to antibiotics, the development of gene cloning and expression vectors, and the modifications of the host organisms.
Archaea ; genetics ; Cloning, Molecular ; Genetic Vectors ; Transformation, Genetic

The consecutive monoculture obstacle is a major problem in the field of Rehmannia glutinosa( R. glutinosa),has severely declined the yield and quality of R. glutinosa. Here,using hi TAIL-PCR and RACE techniques,we have cloned the full-length transcript( 1 573 bp) of Unigene 29334＿All screened by DGE as a consecutive monoculture obstacle response gene of R. glutinosa. Based on ORF Finder prediction,all ORFs detected in the full-length transcript were less than 300 nt,which suggested that the above transcript was confirmed to be a long non-coding RNA( LncRNA). With alignment in R. glutinosa transcriptome,this LncRNA was partially homologous to alanine glyoxylate transaminase 2 gene( Rg AGT2),which was named LncRNA-RgATG2. To further explore the function of LncRNA-RgAGT2,we have examined expression patterns of LncRNA-RgAGT2 and Rg AGT2 at five critical development stages( seedling,elongation,pre-expanding,mid-expanding,late-expanding) in the first and second year replanting of R. glutinosa,respectively. The results indicated that LncRNA-RgAGT2,as a potential regulator,is possible to play a vital role in Rg AGT2 expression regulation. Meanwhile,LncRNA-RgAGT2 has presented significant variation in all development stages of R. glutinosa,which could be used as a " diagnostic label" to assess consecutive monoculture obstacle. This study,for the first time,showed that LncRNA was responsible for the response and regulation of consecutive monoculture obstacle,which would be a powerful supplement to reveal the molecular mechanisms of consecutive monoculture obstacle of R. glutinosa.
Cloning, Molecular ; Gene Expression ; RNA, Long Noncoding ; Rehmannia ; Transcriptome