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Showing 3 results for Gene Cloning
Volume 9, Issue 2 (9-2018)
Abstract
Aims: Antioxidants in sesame oil, including tocopherols and sesamin have greatly increased the shelf life of it against heat. Following the increase in the expression of the cytochrome P450 enzyme encoder (CYP81Q1), the content of sesame is increased in different stages of development of sesame seeds. The aim of this study was cloning, sequencing, and bioinformatics study of CYP81Q1 gene of Iranian sesame (Seamum indicum L.) cultivar.
Materials and Methods: In the present experimental research, DNA was extracted from leaves and stems of Karaj1 sesame cultivar and the target gene was amplified by PCR. Gene was cloned in binary vector pBI121 and confirmed by 3 methods, including enzymatic digestion, PCR, and sequencing. Then bioinformatics characterization of this gene was studied and the Ramachandran plot was drawn on the three-dimensional structure of the gene.
Findings: Cloning was confirmed. DNA sequencing results confirmed the cloned segment. Molecular weight and predicted isoelectric point of the protein were 57021.3 Dalton and 8.46, respectively. The three-dimensional structure of the protein had a good stroke chain. The sequencing result of this gene showed a difference in the 23 nucleotides of this gene in sesame seeds of Karaj 1 (access number KP771974.1) with a reported sequence in the NCBI gene bank (access number AB194714.1), which resulted in the sequencing of the CYP81Q1 gene in Iranian sesame (Karaj 1) at this database.
Conclusion: Based on nucleotide sequencing, the target gene has 1521 base pairs, and differs from 23 nucleotides with the sample registered at the NCBI World Bank. This gene encodes a protein length of 506 amino acids. This protein is very similar with the registered protein in NCBI.
Najme Berenjkar, Mohammad Reza Kalbasi, Saman Hoseinkhani,
Volume 10, Issue 2 (4-2021)
Abstract
Piscidin has a wide range in killing microorganisms including bacteria, fungi, viruses, parasites and has strong anti-tumor activity and plays a role in increasing innate immunity and also does not provide resistance against bacteria; Therefore, it is of great importance in aquaculture. In this study, piscidin gene of Sparus aurata in vector pTZ57R / T was cloned. In this research, ligation product was transferred to component cell of E. coli DH5α strain. Plasmid extraction was performed from single colonies observed in ampicillin plate. Confirmation of the accuracy of single colonies grown in this research was performed by direct PCR and sequencing. The amplified cDNA fragment of the gilthead seabream piscidin gene consists of 310 nucleotides and 57 amino acids. The results of this research show that piscidin gene has been successfully cloned in pTZ57R / T vector. Comparison of nucleotide sequence of piscidin gene in this study showed high similarity with piscidin 5 of Morone chrysops. The comparison of the amino acid sequence of signal peptide piscidin is quite similar to Dicentracin-like of that species registered in the genebank, and mature peptide piscidin sequence is similar in only three amino acids to Pleorocidin-like of Poesila farmosa and Dicentracin-like of Sphaeramia orbicularis. This study could be a step towards further studies of piscidin peptide.
Volume 15, Issue 81 (11-2018)
Abstract
Animal chymosin due to the high quality of cheese texture and flavor is the most effective enzyme for cheese production. Due to the increasing demand for this enzyme, in addition to the plant and microbial sources, recombinant sources should be considered for chymosin production. In this study, in order to produce recombinant chymosin, as well as improving the expression of bovine chymosin gene in yeast, the sequence of the chymosin A gene was optimized and synthesized based on the codon usage of the Pichia pastoris and was cloned in an appropriate expression vector. The codon adaptation index (CAI) increased from 0.59 to 0.72 after codon optimization. There were 41 yeast rare codons (with frequency of less than 10%) in the bovine chymosin A gene, whereas no rare codons in the codon optimized sequence. In order to transfer the resynthesized chymosin A gene to the yeast expression vector pPIC9, this gene was proliferated using the specific primers and was cloned in the PTG-19 vector. Recombinant (PTG19-chymA) colonies were selected by the screening of white-blue colonies method. The resynthesized chymosin A gene was then cut from PTG19-chymA vector using the restriction enzymes Not I and EcoR I, cloned into the pPIC9 vector and was transformed into DH5α strain of E. coli. Colonies containing recombinant vector (pPIC9-chymA) were identified and selected using colony-PCR technique. The recombinant nature and correct insertion of resynthesized chymosin A gene in the yeast expression vector pPIC9 were confirmed by plasmid extraction and its digestion with BamH I enzyme.
