Cloning And Functional Analysis Of Maize Phospholipase D Gene Family

Phospholipase D(PLD)is a class of phosphatidylcholine phosphatidylhydrolase,which specifically hydrolyze the 4-position phospholipid bond of the phospholipid molecules,producing phosphatidic acid(PA),which plays important roles in growth,development,and stress response of plant,as a class of lipid second messengers.Genome-wide identification of the PLD gene family has been reported only in several model plants.A lot of repeat and rearranged sequences in the maize genome make it much more complex.This might lead to evolutional and functional diversification among the members of the ZmPLD gene family.However,only a few literatures are available for cloning and functional elucidation of several members of the ZmPLD gene family.In this study,genome-wide identification for ZmPLD gene family was carried out according to homologous alignment and conserved domain analysis.Biofinformatics analysis was conducted for chromosome localization,phylogenetic reslationship,cis-acting elements,gene structure,and expression pattern of the members of the ZmPLD gene family.Real-time quantitative PCR was used to elucidate the expression charactristics of these members under the abiotc stresses and hormone induction,in order to analyze the molecular mechanism of abiotic tolerance in maize.The main results are as follows:(1)By homologous alignment and conserved domain analysis,thirteen members of the ZmPLD gene family were identified from the maize genome.They distribute randomly on seven of the tenchromosomes.According to the homology of their putative proteins with the PLD proteins of Arabidopsis and rice,eight,one,three and one of these thirteen members were classed into subfamilies ?,?,? and ? respectively.Zero,one,two,three and nine introns were identified from these thirteen members,respectively,indicating their diversity of gene structure.Of the thirteen putative proteins,twelve contain the PX-PH and the C2 domains,except ZmPLD? contains the HKD and the C2 domains.(2)By using PlantCARE software,multiple cis-acting elements responsive to hormone induction of abscisic acid,methyl jasmonate,and gibberellin were identified from upstream of the encoding sequences of the thirteen ZmPLD genes,indicating that these genes are probably regulated by groth,development and abiotic stress,and involved in different signaling pathways.(3)The expression patterns of the ZmPLD family members in different tissues and organs and at different development stages were analyzed on the basis of the expression data in the related databases.The expression of the ZmPLD?1 and 2 genes was persistently high,the expression of the ZmPLD a3,4,6 and 8 genes was persistently low,except the ZmPLD?3 and 4 genes had an expression peak in the anther.The other seven genes had different expression patterns in different organs or at different stages.This result indicated that the members of the ZmPLD gene family play different functions in the different stages of maize growth and development.(4)By analysis of quantitative PCR,the expression of all the ZmPLD family members was upregulated in shoot and root of maize seedlings under the stress of 16%polyethylene glycol 6000,except the expression of the ZmPLDa8 gene was downregulated in the root.Under the stress of 250 mmol/L NaCl,the expression of the ZmPLDa2,6 and 7 genes had an upregulated peak in the shoot,while the ZmPLDa6 and 7 genes showed an upregulated peak in the root.The expression of the other members showed no obvious variation.Under the stress of 4 C temperature,the expression of most members were significantly downregulated,whereas the expression of the ZmPLDa7 gene was significantly upregulated in the root.(5)In response to the induction of 100 ?mol/L abscisic acid,the expression of most members was significantly upregulated in the shoot,except the expression of the ZmPLDal gene showed no response and the expression of the ZmPLD? gene was downregulated.The expression of the ZmPLDal,3,6,7,8,and the ZmPLD? gene was upregulated in the root,and the expression of the ZmPLD?2,4,5 genes,the ZmPLD?1,2,3 genes,and the ZmPLD? gene was downregulated in the root.In response to the induction of 100 ?mol/L methyl jasmonate,the expression of most membes was upregulated to different degress in the shoot and root,and some of them had expressin level ten time more than the control,whereas the expression of the ZmPLDa7 gene was downregulated in the shoot.In response to the induction of 100?mol/L gibberellic acid,the expression of most members showed no regular response,whereas the expression of the ZmPLDa4,6 genes,and the ZmPLD?1 gene in the shoot,and the ZmPLD?7,the ZmPLD?,and the ZmPLD?1 genes in the root showed an obvious peak.The above results elucidated the real presence of the thirteen members of the ZmPLD gene family,and indicated obvious diversity of gene structure and expression pattern among these members.They probably play different functions in growth,development and stress response of maize.