Study On Prolyl Oligopeptide(POP) Genes Encoding A Key Enzyme Of Cyclic Peptide Toxin Biosynthesis In Amanita Exilialis

Amanita exitialis Zhu L.Yang & T.H.Li is the top killer of mushroom poisonings in Guangdong Province,the toxic cyclopeptides produced by the species not only are lethal,but also has broad application prospects in new drug researches.Previous studies have shown that Prolyl oligopeptidase(POP)is known as the key enzyme in the biosynthesis of the Amanita cyclopeptides.However,few studies have been reported about this key enzyme,and the POPs in the lethal Amanita species endemic in C hina have not been studied.In this study,we cloned the gene sequences of POPA(named Ae POPA)and POPB(named Ae POP B)in A.exitialis,and analyzed the characteristics of predicted amino acid.Relationship of AePOPB gene expression patterns and α-amanitin accumulation in different growth periods and parts of A.exitialis were further analysed.Besides,the copy numbers of AePOPB in A.exitialis were also investigated.The main results were as follows:1.cDNA of Ae POPA and AePOPB were firstly cloned and analysed.The ir lengths are 2286 bp and 2193 bp,and encoding 761 and 730 amino acids respectively.Usingc DNA sequences of Ae POPA and AePOPB as the reference to design primers and amplify the genomic DNA of A.exitialis,the full-length sequences of Ae POPA(3135bp)and AePOPB(3144bp)were obtained.c DNAand the full length DN A sequences were compared the result showed that both of Ae POPA and AePOPB contained 18 exons and 17 introns.Secondary structure analysis showed that both Ae POPA and AePOPB proteins have single-type Peptidase＿S9＿N and Peptidase＿S9 structure.The hydrophobic or hydrophilic analysis indicated that the hydrophilic amino acids of Ae POPA and AePOPB are equal distribution from the N to C,and most amino acids belonged to the hydrophilic amino acids,so Ae POPA and AePOPB proteins vest to hydrophilic proteins.2.Comparing Ae POPin A.exitialis with those of other species downloaded in the NCBI database,the results revealed that protein sequencecoded by AePOPBfrom A.exitialis are highly homologous(97%)with the protein sequencecoded by Ab POPBfrom A.bisporigera.This two species clustered to one group by p hylogenetic analysis.low sequence similarity existed in G.marginata and C.lacteal,76% and 57% respectively.The protein sequence of Ae POPA from A.exitialisare highly homologous(95%)withprotein sequenceof Ab POPA from A.bisporigera.The results showed that Prolyl O ligopeptide and their genes Ae POPA and AePOPB were highly conservative in the lethal species of genus Amanita.3.The expression stability of five internal genes: 60 s,GAPDH,NADH,RPB2 andβ-actin in different growth periods and fruitbody parts of A.exitialiswere tested,and the results shown that β-actin expression was the most stable gene in the A.exitialis,and selected as the best reference gene for real-time quantitative PC R.Expression levels of AePOPB and Ae α-AMA were analysed using the q RT-PCR in four different growth periods and three different parts of A.exitialis,and the results indicated that AePOPB and Aeα-AMA showed high expression level at elongation stage butthe lowest expression level in volva.In addition,AePOPB and Ae α-AMA expression in the same tissues in different periods have high consistency.Contents of α-amanitin in different periods and parts were also detected in this study.The results showed that α-amanitin content instipe is highest at early elongation stage.At the early elongation stage and late elongation stage,α-amanitin content in pileus is highest.The results showed that high expression levels of the toxin synthesis related genes AePOPB and Aeα-AMAwere in the early growth period of A.exitialis,and highcontents of thetoxin were in the middle growth period;however,the gene expression levels and the toxin contents have a high degree of consistencyin different parts of A.exitialis.4.The enzyme loci for AePOPB gene was analysed,and digestion enzyme Pst I was selected to digestion genome DNA.Using southern blot technique,AePOPB gene in A.exitialis was two copies,which suggested Ae POP B gene existed with the form of double copies in the genome,and each one more copy number could accelerate biosynthesis metabolism.On the whole,this research not only definited the total length and structure of the gene Ae POP in A.exitialis,but also specified the role of AePOPB genes in amanitin peptide synthesis,these results have laid a solid foundation for later study in amanitin peptide synthesis pathway and its biological regulation mechanism.