Cloning And Functional Analysis Of Diacylgycerol Acyltransferase Gene(PfDGAT) In Perilla Frutescens

Perilla(Perilla frutescens(L.)Britt.)is an important edible-medicinal oil crop,with its seed containing 45-55% oil.Of perilla seed oil,?-linolenic acid(ALA,18:3?9,12,13)accounts for 55%-65%?ALA,one kind of health-promoting fatty acids,functions importantly in human brain development and cardiovascular system healthcare.However,the mechanism underlying ALA biosynthesis and perilla oil accumulation is still unclear.In this paper,molecular cloning technique was employed to isolate diacylglycerol acyltransferase(DGAT)genes(Pf DGAT1 and Pf DGAT2),the key genes responsible for triacylglycerol(TAG)biosynthesis,from developing perilla seeds.The expression profiles of Pf DGAT1 and Pf DGAT2 were examined in various organs/tissues and different seed development stages of perilla by quantitative PCR.Expression vectors of Pf DGAT1 and Pf DGAT2 were separately constructed,and subsequently transformed into yeast(Saccharomyces cerevisiae)and two kinds of tobacco(Nicotiana tobaccum and N.benthamiana),respectively,to elucidate the biological functions of these two genes.The present study provides the new knowledge for understanding of perilla seed oil biosynthesis and regulation mechanism,and also the scientific basis for genetic improvements of oil yield and quality in oilseedsThe main findings are described as the followings:1.The transcripts of Pf DGAT1 and Pf DGAT2 were identified by screening the perilla seed transcriptome data generated previously in our lab.q RT-PCR analysis showed that Pf DGAT1 and Pf DGAT2 expressed in all perilla organs/tissues tested,but their expression levels were different in these tissues.Different expression profiles of Pf DGAT1 and Pf DGAT2 in developing seeds were also detected between two perilla varieties,‘Jinzisu 1' and ‘Bingzisu 1',with the peak level in seed at 20 days after flowering(DAF)and 10 DAF,respectively,for ‘Jinzisu 1' and ‘Bingzisu 1'.2.The c DNA clones of Pf DGAT1 and Pf DGAT2 genes were successfully isolated from the perilla developing seeds of Jinzisu 1.The Pf DGAT1 c DNA sequence was 1964 bp in length,which contains 1605 bp of open reading frame(ORF)encoding 534 amino acid residues.The Pf DGAT2 c DNA length was 1249 bp,which contains an ORF of 990 bp encoding 329 amino acid residues.Functional structure domain prediction indicated that Pf DGAT1 and Pf DGAT2 contain the domains of PLN02401(diacylglycerol o-acyltransferase)and PLN02783(diacylglycerol o-acyltransferase)belonging to the super families of MBOAT and LPLAT,respectively.Multiple sequence alignments and phylogenetic analysis revealed that Pf DGAT1 and Pf DGAT2 proteins had the close genetic relationship with their homologs from Sesamum indicum and Nicotiana tabacum,respectively.3.The yeast expression vector p YES2.0 containing Pf DGAT1 or Pf DGAT2 ORF was successfully constructed,and then transferred into the yeast(Saccharomyces cerevisiae)mutant strain H1246 deficient in TAG biosynthesis in order to perform functional complementation assay.Nile red staining of oil bodies and TLC analysis showed that the overexpression of either Pf DGAT1 or Pf DGAT2 gene could restore the TAG biosynthesis and accumulation in the yeast mutant H1246,indicating that the proteins encoded by Pf DGAT1 and Pf DGAT2 genes had the DGAT enzyme activity.The total oil content in the transgenic yeast of Pf DGAT1 or Pf DGAT2 was significantly increased.GC analysis on fatty acid profiles revealed that the contents of C16:1 and C18:0 in the transgenic yeast were significantly lower than those in the control strain,accompanied by the great increase of C16:0 and C18:1 levels in the transgenic yeast.4.The plant overexpression vector p CAMBIA1303+Pf DGAT1 and p CAMBIA1303+Pf DGAT2 were successfully constructed,and consequently transformed into Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration to transiently express the target gene.The total oil content and fatty acid profiles in the tobacco leaves were determined,showing the leaves transiently-expressing either Pf DGAT1 or Pf DGAT2 accumulated much high oils compared to the controls.In the target gene-expressed leaves,both C18:1 and C18:3 enhanced significantly,whereas C16:0,C18:0 and C18:2 largely reduced.Furthermore,the two target genes were separately introduced into common tobacco(N.tobaccum,variety:Sumsun NN,SNN)by Agrobacterium-mediated leaf disc transformation.The positive transgenic tobacco plants were obtained by screening and identification.In summary,Pf DGAT1 and Pf DGAT2 c DNA clones were isolated from perilla developing seeds and characterized.The Pf DGAT1 and Pf DGAT2 proteins have high enzymatic activities of DGAT.These two genes could be used in genetic engineering to improve oil yields and quality in oilseeds and plant vegetable organs.