Identification Cloning And Functional Analysis Of Diacylglycerol Acyltransferase Genes In Maize

Diacylglycerol acyltransferase(DGAT)catalyzes the only rate-limiting step in pathway of plant oil(triglycerol-TAG)biosynthesis,and also involves in plant growth and development.In this study,the genome-wide identification and functional characterization of maize DGAT gene family were conducted via bioinformatics and other strategies,which include:(1)The maize DGAT gene family was screened and identified at the whole genome level,and comprehensive bioinformatics analysises were performed.(2)Expression of maize DGAT gene family in response to abiotic stress.(3)The full-length cloning of two DGAT genes of maize and their enzymatic function verification.The major results are as follows:1.Five maize DGAT gene family members were identified from maize genome database,including twotype-I DGAT-ZmDGAT1.1 and ZmDGAT1.2,two type II DGAT-ZmDGAT2.1 and ZmDGAT2.2,and one type DGAT-ZmDGAT3.The DGAT1 and DGAT2 families of maize contain multiple transmembrane domains,while the maize DGAT3 does not contain the transmembrane domain.2.The results of phylogenetic analysis showed that the protein sequences of DGAT1,DGAT2 and DGAT3 were clustered into the same cluster with other-type-I,II and III homologous,respectively.Among different species,DGAT proteins were were further divided as algae,mosses,monocotyledon and dicotyledon.The maize DGAT proteins were classified into monocotyledon group,and exhibited close genetic relationship with sorghum.In the process of evolution,the length and number of introns and exonsof the DGAT orthologs from algae,monocotyledon and dicotyledon went through certain degrees of increase or decrease.3.The conserved functional domains analysis revealed that the protein encoded by ZmDGAT1 contained two MBOAT functional domains(PF03062,Membrane bound O-acyl transferase)and and ARE1 regulatory elements associated with lipid accumulation(alkane-responsive element one).Two maize DGAT2 encoding protein contains the LPAT(Lysophospholipid acyltransferase)conserved domain(also known as MBOAT2)and the membrane-bound protein-like domain of the lysophosphatidyltransferase gene family.The functional domains of Zm DGAT1 and ZmDGAT2 are concentrated at the N-terminus of the protein,while the maize DGAT3-encoded protein contains the cell division class protein ZapA inDGAT3 and the ferredoxin domain similar to ferredoxin / thioredoxin.4.Gene expression analysis showed that the DGAT1 and DGAT2 genes in maize roots and leaf tissues were up-regulated under various abiotic stress conditions,and the expression of maize DGAT genes varied at different time points and different tissues.The different expression pattern implied that the maize DGAT gene family members respond to a variety of abiotic stresses and might play different roles in response to various abiotic stresses.5.The coding region of two ZmDGAT2 gene were cloned into the yeast expression vector pYES2.1,and the validated expression vector was transformed into the yeast lipid mutant H1246 strain for in vitro enzyme activity analysis.The results showed that the expression of DGAT2 could restore the phenotype of yeast mutant,indicating that the protein encoded by maize DGAT2 genes had the enzymatic function of lipid synthesis.