Identification And Functional Analysis Of Type-2 DGAT Homologue Gene From The Green Microalgae Haematococcus Pluvialias

Microalgae,a photosynthetic autotrophic of lower aquatic plant,have the advantages of fast reproduction,short growth cycle and high oil and protein content,so it is considered as the "Blue granary" of high-quality biological resources,and has been widely used in functional agriculture,bioenergy,environmental governance and other fields.Astaxanthin,known as “super vitamin E”,is of great commercial interest due to its high anti-oxidant activity,crucial biological function and a variety of usage such as aquaculture,food industry,health care,cosmetics,health products and other fields.The unicellular green alga Haematococcus pluvialis can produce losts of astaxanthin(up to 3%-4% of the dry weight of cells)under stress conditions such as high temperature,high light and nutrient starvation and is the best source of natural astaxanthin in the world.It also represents a potential source of TAG,since a considerable increase in TAG content accompanies the accumulation of AST.Interestingly,most of AST in H.pluvialis is in the ester form(more than 95%)and is stored in oil bodies.Although the exact mechanisms behind stress-induced TAG and AST accumulation in H.pluvialis are not well understood,several lines of evidence have suggested that(1)the biosynthesis pathways of both compounds appears to be linked through the regulation of TAG biosynthetic enzymes and the accumulation of AST appears to be dependent on the accumulation of TAG,(2)the key rate limiting enzyme diacylglyceryltransferase(DGAT)in TAG synthesis was highly expressed in the above induction process,suggesting that it was involved in the above physiological process,(3)DGAT may be a bifunctional enzyme that catalyzes TAG synthesis and astaxanidin esterification simultaneously in H.pluvialis.Although the function of DGAT has been studied in a large number of higher plants and some microalgae,the identification and function of the DGAT gene family in H.pluvialis have not been reported.Therefore,in this study,the type 2 DGAT family was firstly identified and analyzed based on transcriptome data;Then combined with the content changes of astaxanthin and lipids under stress and the expression profiles of DGAT family members of type 2 it was speculated that Hp DGAT2-2 might have strong activity;Finally,the full length of Hp DGAT2-2 gene was obtained by molecular cloning.The ability of TAG synthesis was verified by yeast system,and the potential of Hp DGAT2-2 in regulating oil synthesis and fatty acid content changes in higher plants was explored by tobacco transformation system.The main results of this study are as follows:1.Five DGAT2 genes(named Hp DGAT2-1,Hp DGAT2-2,Hp DGAT2-3,Hp DGAT2-4 and Hp DGAT2-5)were predicted from the transcription data of H.pluvialis by local blast method based on DGAT2 sequence in A.thaliana and C.reinhardtii.The physical and chemical properties,advanced structure and phylogenetic relationship were analyzed by bioinformatics.The conserved domain analysis showed that all the five Hp DGAT2 genes contained the conserved functional domain LPLAT,indicating that their belonging to the DGAT2 family.Multiple sequence alignment and phylogenetic analysis indicated that Hp DGAT2-2 had a high similarity with the reported gene sequences of Cr DGAT2 B from C.reinhardtii and Cz DGAT2 E from C.zofingiensis with a strong lipid synthesis capacity,which implied that Hp DGAT2-2might also have a strong DGAT activity.2.High light and nitrogen deficiency can effectively improve the contents of astaxanthin and lipids in H.pluvialis.The biomass,astaxanthin,total lipid content and the expression of five target genes under five treatment conditions(HLB,HLW,1/4N,HLW-1/4N,and HLB-1/4N)were analyzed.The results showed that the production of astaxanthin and total lipid in H.pluvialis significantly increased under five stresses condition in the order of HLB-1/4N > HLW-1/4N > HLB > 1/4N > HLW.In addition,the transcriptional expression level of five Hp DGAT2 genes was increased with distinct manners.Specific,the Hp DGAT2-1,Hp DGAT2-2 and Hp DGAT2-4 genes expression gradually increased with the extension of stress time,but only Hp DGAT2-2 gene had extremely high expression level under among five stress conditions.3.In order to further verify the function of the Hp DGAT2-2 gene,a full-length c DNA sequences encoding a putative DGAT2(Hp DGAT2-2)was obtained from H.pluvialis.It contained an open reading frame(ORF)of 1,017-bp encoding a protein of 338 amino acid residues.Hp DGAT2-2 activity was confirmed in the TAG deficient yeast strain(H1246)by restoring its ability to produce TAG.Upon expression of Hp DGAT2-2,C16:0 and C18:1 fatty acid contents were 190.2% and 132.4% higher respectively than that of the H1246 strain.In addition,over-expression of Hae DGAT2 E in transgenic Nicotiana Benthamiana resulted in increased contents of total lipids(134%),C16:0(113.5%)and C18:1(234.5%).In summary,the DGAT2 family in H.pluvialis was firstly identified and sequences analyzed.The Hp DGAT2-2 gene was cloned and the ability of TAG biosynthesis was verified in TAG-deficient yeast strain.Moreover,overexpression of Hp DGAT2-2 in higher plants(tobacco)could increase the TAG content and improve the fatty acid composition.This study not only provided novel alternative DGAT candidate gene for improving oil production by gene editing method in crops,but also laid the foundation for the molecular mechanisms of astaxanthin esterification in H.pluvialis.