تراريزش برنج با ژن مانيتول-1- فسفات دهيدروژناز ﴿توليد كننده مانيتول﴾ به منظور ايجاد تنظيم اسمزي توسط نانو ذرات طلا

چكيده انگليسي :

Transformation of Rice Using Mannitol 1 phosphate Dehydrogenase Gene Mannitol Synthesizer in Order to Create Osmoic Adjustment by Nano Gold Particle Sakineh Mehrani E mail s mehrani@yahoo com 2009 5 19 College of Agriculture Isfahan University of Technology Isfahan 84156 83111 Iran Degree M Sc Language Farsi Supervisor Dr A M Rezaie Supervisor Dr S E Mortazavi Mortazavi se @yahoo com Abstract In order to obtain marker free transgenic rice with improved osmotic stress resistance expression vector named as pABRII MTLDL contains mtld gene and lacks of plant selectable marker gene hph were constructed from pCabMTLDII and pTRA132 to be used in co transformation technique with another plasmid contained hygromycine phosphotransferase gene as plant selectable marker In co transformation technique marker gene and genes of interest are placed on separated DNA molecules and introduced into plant genome as unlinked fragments then the non selectable genes from the marker gene in the progeny are segregated through conventional genetics In this way it would be possible to achieve marker free transgenic plant during the generations of segregation The digestion of pCabMTLDII a 7 7 kb plasmid which contains both mtld and hph genes with the EcoRI and BamHI enzymes resulted in a 1 8 kb fragment which contained the entire E coli coding region for the mt1d structural gene together with nopaline synthease terminator This 1 8 kilobase fragment was then inserted into the EcoRI and BamHI restriction sites of the modified pTRA132 a 4 4 kb plasmid which contains only hph gene in which the hph coding sequence and nopaline synthease terminator had been deleted The cloning of the fragment into the modified vector pTRA132 resulted in a plasmid designated pABRII MTLD The recovery of these 3 kb and 1 8 kb fragment from agarose gel was done by High Pure PCR Product Purification Kit and the ligation of these fragment was done by T4 ligase enzyme using Rapid DNA Ligation Kit This resulted plasmid pABRIIMTLD was about 4 9 kb Mtld gene in the resulted cassette pABRII MTLD confers elevated tolerance against drought and salinity stresses and it is controlled by mosaic virus 35S promoter The plasmid was analyzed by digestion with BamHI EcoRI HindIII and pstI enzymes and PCR analysis in which accuracy of construction were confirmed Then plasmid pABRIIMTLD with pTRA132 containing hph gene in ratio of 1 1 introduced into high cell dividing and regeneration ability embryogenic calli derived from the mature seeds of a rice variety Hashemi by biolistic transformation method About 100 small and globular embryogenic calli were arranged on the center of petridishes and the explant of each petri was shot twice Then selection of transformed cell was carried out on callus induction and also regeneration medium using 50 mg 1 hygromycin B as antibiotic Transformed cells which contain plant selectable marker gene can survive on medium containing antibiotic Putatively transformed cell clusters were identified from the bombarded tissues after 3 rounds selection every 14 21 days on callus induction N6 medium containing 50 mg 1 hygromycinB After 6 8 weeks selection hygromycin resistant calli were transfered to regeneration medium and regenerated on MS medium with 50 mg 1 hygromycinB Several cotransgenic rice plant containing both the mtld gene and the marker gene were regenerated and the integration of both transgenes in the transgenic rice plants was confirmed by PCR Also PCR analysis showed that some of regenerated plant only had plant selectable marker gene hph in their genome This research can confirm the efficiency of cotransformation of rice embryogenic calli with two genes on different plasmid and using biolistic transformation method Key Words Co transformation rice cv Hashemi mtld gene hph gene Biolistic pdfMachine A pdf writer that produces quality PDF files with ease Produce quality PDF files in seconds and preserve the integrity of your original documents Compatible acros

مهراني،سكينه

تراريزش برنج با ژن مانيتول-1- فسفات دهيدروژناز ﴿توليد كننده مانيتول﴾ به منظور ايجاد تنظيم اسمزي توسط نانو ذرات طلا

برنج رقم هاشمي , ژن mtlD , ژن hnh , بايوليستيك