Identification And Potential Applicaion Of A ?3-fatty Acid Desaturase Gene From Perilla Frutescens(L.)

Perilla frutescens(Labiatae)is a medicinal and edible oil plant.Comparing other terrestrial plants,the content of ?-Linolenic acid(ALA)in it is extremely high.?3-fatty acid desaturase can catalyze linoleic acid forming ALA,which increasing the content of ALA in microorganisms and plants.In this study,we carried out transcriptome sequencing and analysis of the plant's RNA from flowering to seed ripening 7 development periods(2d,6d,10 d,14d,18 d,22d,26d).The prokaryotic expression was carried out to create linolenic acid engineering strains,after PfFAD3 gene was extracted and cloned,which provided the basis for the industrial production of ALA and the improvement of the quality of the oil and other crops.The main contents and results are as follows:1)A total of 64,156 unigenes were obtained by assembling and splicing the 7 library.About 150 unigenes were found associated with the synthesis and accumulation of fatty acids and 15 transcription factors with high expression of FAD3.2)Three omega-3 fatty acid desaturase genes(pfFAD3-1 ~ 3)were extracted from the transcriptome data,and the expression pattern of ?-3FAD with the highest expression was verified by Real-time PCR.The results showed that the expression quantity of pfFAD3 was low in flowers and leaves,mainly expressed in seeds.The expression level increased gradually at the 2nd day of seed development,reached the highest at 18 days and then decreased.The expression trend was close to that of RNAseq.3)The pfFAD3 was cloned from perilla seeds and the pBlunt-pfFAD3 cloning vector was constructed.Using the pMal-c2 X plasmid,we constructed a prokaryotic expression vector of pfFAD3 gene pMal-c2X-pfFAD3.The pfFAD3 gene was introduced into Escherichia coli by heat shock method.After PCR identification and sequencing,the ?-3FAD fusion protein was induced by IPTG.The results showed that the content of linolenic acid in the recombinant engineering bacteria was significantly higher than that of the wild type strain(0.91% higher).4)The study of heterologous expression mechanism showed that the coding parameters of the basil seeds were preferred to use the codon ending in A/T,and Saccharomyces cerevisiae was the appropriate host cell of pfFAD3-1~ 3.