Delta8 desaturase pathway, different from common delta6 desaturase pathway, is an alternate pathway of polyunsaturated fatty acids biosynthesis. Delta8-fatty acid desaturase is one of the key enzymes in delta8 desaturase pathway. Two specific fragments were separately cloned from genomic DNA and cDNA of Euglena gracilis by PCR with the primers designed according to the reported sequence. Comparison of the genomic and cDNA sequences revealed that there wasn't intron in this delta8-fatty acid desaturase gene. This gene has an open reading frame of 1 266 bp that encodes 421 amino acids. It is 6 bp longer than the reported gene sequence, and also showed certain difference from the reported sequence in the N-terminal. The recombinant expression plasmid pYEFD by subcloning delta8-fatty acid desaturase gene into the yeast-E. coli shuttle vector pYES2.0 was constructed and was transformed into the defective mutant INVSc1 of Saccharomyces cerevisiae by electrotransformation. The resulting strain YD8 harboring plasmid pYEFD was selected and was cultured in the induction medium with exogenous substrates omega6-eicosadienoic acid and omega3-eicosatrienoic acid for the expression of delta8-fatty acid desaturase gene. The results indicated that high level expressed As-fatty acid desaturase could convert omega6-eicosadienoic acid and omega3-eicosatrienoic acid to dihomo-gamma-linolenic acid and eicosatetraenoic acid with substrate conversion ratio 31.2% and 46.3%, respectively.
Amino Acid Sequence ; Cloning, Molecular ; Euglena gracilis ; enzymology ; Fatty Acid Desaturases ; biosynthesis ; genetics ; isolation & purification ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism

Online and offline blended learning is the key link to change the teaching mode of higher education. As a new teaching mode, it faces problems such as too much emphasis on students' autonomous learning ability, inadequate supervision of students, and difficulty in building a holistic view of the medical system when applied to medical education. To better promote the reform of blended learning in medical colleges and universities, it is necessary to take the following actions: designing the course content carefully and scientifically arranging the length of online lectures; adopting various ways to follow up in all aspects and building a "whole course" supervision system; always maintaining a holistic view of medicine throughout the blended learning to cultivate comprehensive talents.