Cloning and expression analysis of delta-12 fatty acid desaturase gene in Coix lacryma-jobi L / 药学学报

In this study, the fatty acid desaturase gene FAD2 was cloned from Coix lacryma-jobi L. and its molecular structure and function were studied. The results showed that the full-length cDNA sequence of FAD2 gene was 936 bp encoding 311 amino acid residues. Bioinformatics prediction results showed that the protein encoded by the FAD2 gene was an alkaline hydrophilic unstable protein with a molecular weight of 34.87 kDa. It contained three transmembrane helix domain, and did not contain the signal peptide splicing site, and was most likely to be located in plasmid membrane. Compared with other similar genes in plants, it has only a histidine conserved site, His Box Ⅲ histidine site (HXXHH), suggesting its activity may be reduced. Phylogenetic tree analysis showed that FAD2 was closely related to monocotyledonous plants, especially Maize and Oryza sativa japonica Group, but farther from dicotyledonous plants. Therefore, it was inferred that FAD2 might have similar functions with similar genes in Maize and Oryza sativa japonica Group. In addition, the expression of FAD2 gene could be detected in Coix lacryma-jobi L. with high oil content, but not in low oil content of Coix lacryma-jobi L. In order to clarify the function of FAD2, the gene was heterologously expressed in sporomyces cerevisiae. The results showed that the protein encoded by FAD2 gene did not catalyze the formation of C18∶1 unsaturated fatty acid into C18∶2 unsaturated fatty acid. Therefore, it was speculated that the deletion of histidinine conserved site of FAD2 gene might lead to the decrease of protein activity or even inactivation. This study provides reference value for further understanding the molecular structure characteristics of fatty acid desaturase. At the same time, it laid a foundation for elucidating the biosynthetic pathway of Coix lacryma-jobi L.

Cloning and bioinformatic analysis of the 3-ketoacyl-CoA synthase gene in Coix lacryma-jobi L / 药学学报

As a key gene in the regulation of long-chain fatty acid biosynthesis, 3-ketoacyl-CoA synthase (KCS) plays an important role in the growth and development of Coix lacryma-jobi L. In this study, the KCS gene was cloned from cDNA of Coix lachryma-Jobi L. and bioinformatics analysis was performed. Results showed that the full length KCS gene was 1 548 bp encoding 515 amino acids. Bioinformatics analysis indicated that the gene encoded a 58 608.12 Da protein with an isoelectric point of 9.20 containing two transmembrane helical structure domains and lacking a signal peptide, with a likely subcellular localization in main plastid membranes. The results of multiple sequence comparisons and evolutionary tree analysis revealed that KCS had three identical conserved sequences and was closely related to KCS from monocotyledons such as Sorghum bicolor, Zea mays, Setaria italica, Panicum miliaceum, Oryza brachyantha, Hordeum vulgare, Aegilops tauschii subsp. Tauschii. We speculated that the evolution of the gene was similar among these plants of the same family. In addition, gene expression analysis showed that the KCS gene was significantly different in Coix lacryma-jobi L. isolates having different lipid content. This work will facilitate further study of the regulatory mechanism of this enzyme in fatty acid synthesis.