Functional Research And Sites Analysis Of ApHAL3 Gene In Aureobasidium Pullulans

HAL3 protein family is a kind of highly conservative flavoprotein family.HAL3 protein is a Ppz protein phosphatase inhibitory factor in yeast.The HAL3 proteins in Oryza sativa and Arabidopsis have 4'-Phosphopantothen oylcysteinedecarboxylase(PPCDC)function as same as that the N-terminal domain of the Escherichia coli Dfp protein has PPCDC is a key enzyme in the CoA biosynthetic pathway,and it catalyzes the decarboxylation of(R)-4'-phospho-N-pantothenolycysteine(PPC)to 4'-phosphopantetheine(PP).HAL3 protein also highly relate to salt tolerance.The overexpressions of the HAL3 genes can improve the tolerance of transgenic plants to osmotic stress and salt stress.Aureobasidium pullulans is a fungi that has higher salt tolerance.However,by now little is known about the Aureobasidium pullulans ApHAL3 gene.We studyed the ApHAL3 gene in Aureobasidium pullulans and made sites analysis of the ApHAL3 protein.Alignment analysis revealed that ApHAL3 has a high sequence homology to the N-terminal domain of the Dfp protein in Escherichia coli,AtHAL3a protein in Arabidopsis thaliana and OsHAL3 protein in rice,which have PPC-DC activity.The ApHAL3 protein contains four conserved motifs just as other HAL3 proteins:the substrate binding helix,insertion His motif,PXMNXXMW motif,and substrate recognition clamp.We cloned the ApHAL3 protein cDNA sequence by RT-PCR technology and construction of plasmid vector.Complementation tests demonstrated that ApHAL3 rescued the temperature-sensitive and auxotrophy phenotypes of the Escherichia coli ?dfp mutant strain with PPCDC defect.The results indicate that the ApHAL3 protein has the function of PPCDC.A series of vectors were constructed with point mutations of ApHAL3.Further complementation tests indicated that the conserved Leu40,Trp106,Met120,Asn172,Cys212,Met220 and Ser116Ala117Asn118Ser119 residues of ApHAL3 are key active-site residues.The mutant ApHAL3 His100and Met175 partly reduced its activity because the E.coli Adfp strains with ApHAL3 His100 and Met175 grew slowly at 42?.The conserved Va120,Arg105,Ala170,Leu202,Ala211,Asp214,Gly216 and Asp235 were not important for the ApHAL3 activity as shown in the complementation tests with Escherichia coli Adfp.We used qRT-PCR to analyze the expression of ApHAL3 in Aureobasidium pullulans on treatment with 1 mol/L,1.5 mol/L and 2 mol/L NaCl under 4 h,8 h,12 h,20 h,respectively.The results showed:the expression of ApHAL3 gene was significantly down-regulated when NaCl concentration is 1 mol/L,1.5 mol/L or 2 mol/L under 12 h;ApHAL3 gene expression increases a little when NaCl concentration is 1 mol/L,1.5 mol/L under 20 h,but not very significant.