7677

7149

كارشناسي ارشد

بيوتكنولوژي

اصفهان: دانشگاه صنعتي اصفهان، دانشكده كشاورزي

1391

پانزده،86ص.: مصور،جدول،نمودار

ص.ع.به فارسي و انگليسي

بهرام شريف نبي، فرهاد شكوهي فر

بدرالدين ابراهيم سيد طباطبائي، ضياء الدين بني هاشمي

4/2/92

آقا فخر مير لوحي، مجيد طالبي

مهندسي كشاورزي

ID7149

به فارسي و انگليسي: قابل رويت در نسخه ديجيتالي

Detection amplification and cloning of Tomatinase gene from Fusarium oxysporum f sp melonis Fateme zarandi fatemezarandi@gmail com Bahram sharif nabi Supervisor Sharifnabi20000@yahoo com Farhad shokouihfar Supervisor shokouhifar@um ac ir Department of Agricaltural Biotechnology Isfahan University of Technology Isfahan Iran Degree M Sc Language Persian Date 2013 1 21 Abstract Fungi use different tools to overcome plant defense mechanisms Phytoanticipins are a part of defense materials which are produced in plants to prevent invasion of pathogens Tomatinase is produced and secreted by a range of fungi including some forma specialis of Fusarium oxysporum to degrade phytoanticipins In this study due to agricultural importance of melon crop in the Khorasan provinces Tomatinase presence have been investigated in genome of race 1 and 1 2 of F oxysporum f sp melonis causal agent of Fusarium wilt disease as an economically important disease of melon For this purpose specific primers PSh30 F R based on the F oxysporum f sp lycopersici Tomatinase gene were designed after defining the specific sites in up and down stream of the gene Result revealed the specific primer pair amplified a single 1 1 kb band of the expected size in all tested isolates The segment cloned in pTG19 T vector and transferred to Dh56 Recombinant colonies confirmed by colony PCR technique using the specific primer pair and M13 universal primers A confirmed colony selected for sequencing and Sequencing results showed some variations in Tomatinase sequence of FOM To design an expression construct of Tomatinase the specific primers Psh30 3 F R based on Spe1 and Sac1 restriction site were designed Results showed the specific primer pair amplified single 1056 bp band of Tomatinase carrying the Spe1 and Sac1 sites To express the protein amplified Tomatinase ligated to pET vector and transferred to BL21 This construct pETFOM was sequenced and results revealed that pETFOM construct was designed in correct frame and it doesn t have any stop codon Expression of Tomatinase protein required additional factors such as bacterial strain changing reduction of induction temperature and the other concentration of IPTG The expression of Tomatinase was confirmed with RT PCR So the results based on multiple alignment of Tomatinase showed that FOMTom is a member of glycosyl hydrolases family 10 and is closed to FoTom1 Keywords melon fusarium wilt disease Tomatinase gene detection

بهرام شريف نبي، فرهاد شكوهي فر

بدرالدين ابراهيم سيد طباطبائي، ضياء الدين بني هاشمي

آقا فخر مير لوحي، مجيد طالبي