Isolation And Identification Of Key Genes Involved In The Biosynthesis Of Volatile Organic Sulfur-Containing Compounds (VOSCs) In Tuber Melanosporum

Volatile organic sulfur-containing compounds(VOSCs)are the major contributors to the overall flavor of Tuber melanosporum.In previous work,VOSCs were detected by the addition of methionine to the culture medium of T.melanosporum,and we speculate that VOSCs were synthesized through Ehrlich and Demethiolation pathways.The biosynthesis of VOSCs is the result of changes in gene coordination expression pattern.While molecular mechanism controlling VOSCs biosynthesis in Tuber melanosporum is not yet clear.Based on the knowledge of VOSCs metabolism,we cloned the conserved region of VOSCs synthase genes,screened and analyzed novol genes associated with VOSCs biosynthesis,and attained the rational regulation of VOSCs biosynthesis pathway.Firstly,based on the analysis of highly conserved region,synthase gene homologs in T.melanosporum were cloned and then identified combining with transcriptional expression analysis.Conserved region of AAT1,AAT2,ARO8,ARO8-2,BAT1 and BAT2 encoding amino transferase,PDC1 encoding a-keto acid decarboxylase and ORD1 encoding ornithine decarboxylase,ADH encoding alcohol dehydrogenase and STR3 encoding demethiolase were cloned,which showed 71%(157/221),81%(96/118),78%(169/216),58%(90/154),88%(126/144),71%(169/216),77%(173/225),88%(126/144),57%(45/79)and 87%(201/230)identity to the previously reported homologous genes at amino acid level,respectively.And full-length sequences of AAT1,AAT2,BAT1,BAT2,ARO8-2,PDC1,ORD1,ADH and STR3 are amplified by RACE PCR technology.The addition of methionine resulted in upregulation of aminotransferase genes BAT1,ARO8-2 and a-keto acid decarboxylase gene PDC by 6.09,6.89 and 3.00 fold,while downregulation of branched-chain amino acid aminotransferase gene BAT2,aspartate amino acid aminotransferase genes AAT1 and AAT2,alcohol dehydrogenase ADH and demethiolase gene STR3 by 4.39,4.82,6.04 20.6 and 6.31 fold,respectively.No obvious change was found in aromatic acid aminotransferase gene ARO8 and ornithine decarboxylase gene ORD1.These results suggested that ARO8-2?BAT1 and STR3 may be play main roles during MTL biosynthesis in T.melanosporum.Secondly,forward-and reverse-SSH libraries are constructed and then novel genes associated with VOSCs biosynthesis were screened.1272 and 1133 clones containing differentially expressed genes are acquired from forward-and reverse-SSH library,respectively.And then 92 and 97 positive clones are isolated by reverse-northern blot array from forward-and reverse-SSH library.Cluster analysis showed that these differentially expressed genes in Tuber melanosporum was focused on transcription,translation,protein fate,transport,basal metabolism,stress response and so on.Based on analysis of bioinformation and transcriptional expression,TAR,F-box,PWWP and C6 in transcription,E3 ubiquitin protein ligase gene Ubi346 in protein fate were speculated to be associated with VOSCs synthesis.Finally,E3 ubiquitin protein ligase gene Ubi346 selected from forward cDNA library was overexpressed in T.melanosporum,which lead to the regulation of VOSCs biosynthetic pathway.Full-length DNA sequence of Ubi346(4140 bp)is cloned through a self-formed adaptor PCR,which contained the complete ORF(open reading frame)of 2967 bp predicted by ORF finder.Its expression vector pCAMBIA1302-Ubi was integrated into the chromosal of T.melanosporum with the help of Agrobacterium tumefaciens-Mediated Transformation(ATMT).And then the change in VOSCs biosynthesis was analyzed.The overexpression of Ubi346 significantly increased the biosynthesis of MTL and its derivatives in Demethiolation pathway,while suppressed the biosynthesis of methional and methionol in Ehrlich pathway.This is the first report that overexpression of Ubi346 lead to the direct regulation of VOSCs biosynthesis pathway.