ردیابی همولوگ ژن توماتیناز در ژنوم قارچ Fusarium Oxysporum F.Sp. Melonis

قارچ‌های بیماریزای گیاهی جهت غلبه بر سیستم دفاعی گیاه از ابزار مختلفی بهره می‌گیرند. ترکیبات فیتوآنتیسیپین از جمله مواد دفاعی است که در گیاه جهت جلوگیری از حمله بیمارگرها تولید می‌شود. آنزیم توماتیناز جهت تجزیه فیتوآنتیسیپین و غلبه بر سیستم دفاعی گیاه توسط طیفی از قارچ‌ها از جمله تعدادی از فرم‌های اختصاصی قارچ f. oxysporum تولید می‌شود. با هدف ردیابی توالی کد کننده آنزیم توماتیناز در فرم اختصاصی fusarium oxysporum f. sp. melonis (fom) آغازگرهای اختصاصی بر اساس توالی محافظت شده بالادست و پائین دست توالی کدکننده ژن fotom1 گزارش شده از فرم اختصاصی .fusarium oxysporum f. sp. lycopercisi (fol) طراحی شد. استخراج dna از نژاد یک، شایع در استان خراسان، انجام شد و واکنش تکثیر با استفاده از آغازگرهای اختصاصی صورت گرفت. الگوی الکتروفورزی محصول واکنش حضور یک تک باند در اندازه مورد انتظار را تایید نمود. قطعه تکثیر شده بصورت دو جهته توالی‌یابی شد و با توالی گزارش شده از فرم اختصاصی fol مورد مقایسه قرار گرفت. نتایج توالی یابی با استفاده از برنامه vector nti v11 بر هم منطبق شد و با توالی رفرنس همردیفی انجام شد. نتایج نشان داد در سطح نوکلئوتیدی 14 جهش در توالی شناسائی شده وجود دارد که از این بین 7 جهش در سطح توالی پروتئینی نیز بروز می‌یابند. آنالیز توالی با استفاده از برنامه pfam وجود یک توالی سیگنال پپتید و یک دومین هیدرولازی را در توالی شناسائی شده تایید نمود. این نتایج نشان داد که توالی ردیابی شده میتواند همولوگ ژن توماتیناز در ژنوم فرم اختصاصی fom باشد. این توالی fom-tom نامگذاری شد و در بانک ژن با شماره بازیابی mf178403 ثبت شد. با هدف پیش‌بینی اثر جهش‌ها شناسائی شده در عملکرد ژن توماتیناز آنالیزهای نرم‌افزاری انجام شد که نشان داد جهش‌های رخ داده در توالی دومین هیدرولازی ساختمان پروتئین را تحت تاثیر قرار می‌دهند و می‌توانند در عملکرد ژن تاثیرگذار باشند.

Homology Base Tracking of Tomatinase in the Genome of Fusarium oxysporum f. sp. melonis

Introduction: Phytopathogenic fungi exposes various proteins to overcome plant defense systems. Production of saponins likes αTomatine is one of the tomato preformed defenses barriers which should be detoxicated by the pathogens. It has been revealed before which most of Fusarium species and forma specials could produce tomatinase, a glycosyl hydrolases protein, to deglycosylate αTomatine. Fusarium oxysporium f. sp. melonis (FOM) wildly attached melon cultivars and at the time of this investigation, there was only one report underlining the existence of the gene sequence of tomatinase in the genome of FOM using southern blotting experiment. This study was carried out to track the whole tomatinase gene sequence in the FOM genomic sequence and investigate the probability genetic variation of the gene in the nucleotide and protein sequences.;Materials and Methods: Fusarium oxysporium f. sp. melonis (Fom) race1 have been previously reported in Khorasan, Iran. It was cultured in liquid medium and the mycelia were used for the genomic DNA isolation. Primers were designed based on conserved sequence in upstream and downstream of FoTom1 sequence (AJ012668). PCR was carried out and amplified segments were bidirectional sequenced. The results were then analyzed by Vector NTi software. The sequencing result was aligned with FoTom1 sequence as Refseq and the single nucleotide variations were detected by CLC work bench software. The effects of the mutations on the protein structure were predicted by CLC work bench software.;Results and Discussion: Electrophoresis pattern of PCR products showed a single band of the expected size in the strain FomR1 that was at the same size of the band amplified from FoL genome. The designed primers based on the FoTom1 sequence amplified a specific segment in the Fom genome. Alignment the sequencing results with the FoTom1 from Fusarium oxysporium f. sp. lycopercisi (Fol) in nucleotide level revealed 14 mutations which seven of them were appeared in the protein sequence. Three mutations occurred in the functional domain of the protein. At the position 145, an acidic amino acid with a negative charge was substituted by a polar amino acid. Replacements at positions 218 and 236 were the same in terms of polarity and at position 218; both amino acids have a positive charge.;There were no  introns  in the  coding sequence of FomTom R1 region as same as FoTom1. Pairwise alignment results showed some ansynonyms mutations between two sequences that made some changes in the secondary structure of the translated protein from FomTomR1. The first ansynonym mutation, SNP15 (E→G), inside the signal peptide, converts the alpha helix to a new beta sheet. SNP34 (K→N) and SNP35 (S→N) mutations shortened the alpha helix. The other mutations happened out of the alpha helixes and beta sheets. To predict the effects of the mutations on the FomTomR1function, insilico analyses were carried out. The results revealed that three mutations occurred in the functional domains of tomatinase in Fom. The mutations in the hydrolysis domain may affect the structure of FomTomR1and can be effective in the protein. The presence of different active saponins components in Melon may be an evolutionary reason for some variation in sequence and structure of the FomTomR1 protein in Fom. To prove the differences in the tomatinase function, the interaction of proteins with various types of melon saponins components should be investigated at the future studies.            ;Conclusion: The results showed the tracked sequence could be homolog of the Tomatinse gene in the Fom genome. We named it FomTomR1 and the sequenced was submitted in the Genbank with accession number MF178403. For the future study, the gene influences should be investigated in the pathogenesis of FOM on melon cultivars and it could be considered as a general screening index using heterologous expression of the gene.