Carboxyl CoA ligases(CCLs) is an important branch of adenylate synthetase gene family, which mainly has two-step catalytic reactions. Firstly, in the presence of adenosine triphosphate, it can catalyze the pyrophosphorylation of carboxylateswith diffe-rent structures to form corresponding acyl adenosine monophosphate intermediates. Secondly, adenosine monophosphate was replaced by free electrons in the mercaptan group of enzyme A or other acyl receptors by nucleophilic attack to form thioesters. In this study, on the basis of the transcriptome database of Arnebia euchroma, two genes were selected, named AeCCL5(XP_019237476.1) and AeCCL7(XP_019237476.1). Bioinformatics analysis showed that their relative molecular weights were 60.569 kDa and 60.928 kDa, theoretical PI were 8.59 and 8.92, respectively. They both have transmembrane domains but without signal peptide. By multiple sequence alignment and phylogenetic tree analysis, we found that the similarity between AeCCLs and other plant homologous proteins was not high, and the substrate binding sites of AeCCLs were not highly conserved. The reasons might be that the sequence and structure need to adapt to the changes of new substrates in the process of evolution. In this study, the full-length of AeCCL5 and AecCCL7 were cloned into the expression vector pCDFDuet-1. The proteins of AeCCL5 and AeCCL7 with His-tag were expressed in Escherichia coli. The proteins of AeCCL5 and AeCCL7 were purified by nickel column. In vitro enzymatic reactions proved that both AeCCL5 and AeCCL7 can participate in the upstream phenylpropane pathway of shikonin biosynthesisby catalyzing 4-coumaric acid to produce 4-coumarin-CoA, and then to synthesis p-hydroxybenzoic acid, which is an important precursor of shikonin biosynthesis in A. euchroma.
Boraginaceae/genetics* ; Cloning, Molecular ; Coenzyme A ; Coenzyme A Ligases/genetics* ; Ligases ; Phylogeny

Raspberry ketones have important therapeutic properties such as anti-influenza and prevention of diabetes. In order to obtain raspberry ketone from Chlamydomonas reinhardtii, two enzymes catalyzing the last two steps of raspberry ketone synthesis, i.e. 4-coumaryl-CoA ligase (4CL) and polyketide synthase (PKS1), were fused using a glycine-serine-glycine (GSG) tripeptide linker to construct an expression vector pChla-4CL-PKS1. The fusion gene 4CL-PKS1 driven by a PSAD promoter was transformed into a wild-type (CC125) and a cell wall-deficient C. reinhardtii (CC425) by electroporation. The results showed the recombinant C. reinhardtii strain CC125 and CC425 with 4CL-PKS1 produced raspberry ketone at a level of 6.7 μg/g (fresh weight) and 5.9 μg/g (fresh weight), respectively, both were higher than that of the native raspberry ketone producing plants (2-4 μg/g).
Acyl Coenzyme A ; Butanones ; Chlamydomonas reinhardtii/genetics* ; Ligases ; Polyketide Synthases

4-coumarate coenzyme A ligase is a key enzyme of phenylpropanoid metabolic pathway in higher plant and may regulate the biosynthesis of ferulic acid in Angelica sinensis. In this study, the homology-based cloning and rapid amplification of cDNA ends (RACE) technique were used to clone a full length cDNA encoding 4-coumarate coenzyme A ligase gene (4CL), and then qRT-PCR was taken for analyzing 4CL gene expression levels in the root, stem and root tissue at different growth stages of seedlings of A. sinensis. The results showed that a full-length 4CL cDNA (1,815 bp) was obtained (GenBank accession number: KT880508) which shares an open reading frame (ORF) of 1 632 bp, encodes 544 amino acid polypeptides. We found 4CL gene was expressed in all tissues including leaf, stem and root of seedlings of A. sinensis. The expressions in the leave and stem were increased significantly with the growth of seedlings of A. sinensis (P < 0.05), while it in the root showed little change. It indicates a time-space pattern of 4CL gene expression in seedlings of A. sinensis. These findings will be useful for establishing an experiment basis for studying the structure and function of 4CL gene and elucidating mechanism of ferulic acid biosynthesis and space-time regulation in A. sinensis.
Amino Acid Sequence ; Angelica sinensis ; genetics ; Base Sequence ; Cloning, Molecular ; Coenzyme A Ligases ; genetics ; DNA, Complementary ; chemistry ; Molecular Sequence Data

Stearoyl-CoAdesaturase-1 (SCD-1) is a key regulator of monounsaturated fatty acid synthesis. It plays a vital role in lipid synthesis and metabolism. Ca²⁺ is an important cation in the body and plays an important role in the organism. The aims of this study were to investigate the correlation of SCD-1 gene overexpression with lipid indexes and calcium ion level. The pcDNA3.1 (+) + SCD-1 +Flag eukaryotic expression vector and cultured duck uterine epithelial cells were co-transfected. The overexpression of SCD-1 gene was measured using the Flag Label Detection Kit. Ca ions and lipid contents were detected through Fluo-3/AM Calcium Ion Fluorescence Labeling method and Lipid Measuring Kit, respectively. SCD-1 gene overexpression was negatively correlated with triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C), and positively correlated with Ca ion, total cholesterol (TC), very low-density lipoprotein cholesterol (VLDL-C) and low density lipoprotein cholesterol (LDL-C) levels. Meanwhile, Ca ion was positively correlated with TG, LDL-C and HDL-C contents, and negatively correlated with TC and VLDL-C levels. Overexpression of SCD-1 gene could regulate Ca ion secretion, as well as lipid synthesis and transport in duck uterine epithelial cells.
Animals ; Calcium ; metabolism ; Coenzyme A Ligases ; genetics ; Ducks ; Epithelial Cells ; chemistry ; enzymology ; Gene Expression ; Ions ; Lipids ; genetics ; Triglycerides ; metabolism

AIMTo investigate the roles of the BGR-like gene in testicular development/spermatogenesis.

METHODSA human testis cDNA microarray was hybridized with probes from human adult testes and embryo testes. The differentially expressed clones were sequenced and analyzed. Expression of the BGR-like gene was analyzed by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSA new gene exhibiting 50-fold difference in expression level between adult and fetal human testes was cloned and named the BGR-like gene. The cDNA consisted of 2500 nucleotides and had an open reading frame of 1437 nucleotides encoding a putative protein of 497 amino acid residues. Homologous comparison showed that the BGR-like gene was a new alternative splicing variant of the BGR gene and had sequence homology with the bubblegum gene of human, mouse, rat and Drosophila. Protein motif analysis of the BGR-like gene revealed that it contained a conserved adenosine monophosphate (AMP)-binding domain and a fatty acyl-CoA synthetase signature motif which existed in all acyl-CoA synthetases. The BGR-like gene transcript was imperceptibly expressed in human fetal testes, highly in human adult testes and moderately in elderly testes and human Leydig cells. RT-PCR-based tissue distribution experiments showed that the BGR-like gene was exclusively expressed in testes and was a testes-specific isoform of the BGR gene. A BGR-like gene transcript was not detected in some azoospermic testes.

CONCLUSIONThe BGR-like gene may play an important role in spermatogenesis/testicular development and may be correlated with male infertility.

Adult ; Aged ; Alternative Splicing ; genetics ; Amino Acid Sequence ; Base Sequence ; Coenzyme A Ligases ; genetics ; Drosophila Proteins ; genetics ; Gene Expression Regulation ; Humans ; Infertility, Male ; genetics ; Leydig Cells ; metabolism ; Male ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Spermatogenesis ; genetics ; Testis ; metabolism

OBJECTIVETo study the developmental phase on the growth and active compounds in Scutellaria baicalensis.

METHODSeeds of wild plants were collected from Laiwu and sowed in Fangshan (Beijing) and Laiwu (Shandong). Samples of aerial and underground parts were collected in five growth periods of sprouts, seedlings, flowering, seed drop and withered periods respectively. The length of taproot, fresh weight of root, diameter of taproot and the length of stem were determined. The content of active compounds and total flavonoids were determined by HPLC and ultraviolet spectrophotometry respectively. The transcripted level of PAL1, PAL2, PAL3, C4H, 4CL, CHS, GUS and UBGAT were analyzed with RT-PCR.

RESULTThe results showed that the aerial part of S. baicalensis grew quickly before flowering stage, and the underground part grew mostly between the periods of flowering and withered. In the whole growing developmental periods, the content of total flavonoids was not changed significantly, the content of baicalin was increased gradually and the content of baicalein was decreased gradually. Expression level of PAL and 4CL was the highest in withered period, CHS was increased between flowering and seed drop and decreased in withered period.

CONCLUSIONSeedlings and withered periods may be the key phase affecting the growth and active compounds in S. baicalensis.

Acyltransferases ; genetics ; metabolism ; Chromatography, High Pressure Liquid ; Coenzyme A Ligases ; genetics ; metabolism ; Flavonoids ; analysis ; metabolism ; Flowers ; genetics ; growth & development ; metabolism ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Glucuronidase ; genetics ; metabolism ; Glucuronosyltransferase ; genetics ; metabolism ; Phenylalanine Ammonia-Lyase ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; genetics ; growth & development ; metabolism ; Plant Stems ; genetics ; growth & development ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Scutellaria baicalensis ; genetics ; growth & development ; metabolism ; Seedlings ; genetics ; growth & development ; metabolism ; Spectrophotometry, Ultraviolet ; Time Factors ; Trans-Cinnamate 4-Monooxygenase ; genetics ; metabolism