انتقال ژن شبه تئوماتين TLP

چكيده انگليسي :

Thaumatin Like TLP 3 Gene Transformation into Tobacco Kamran Safavi K safavi@cc iut ac ir 4 3 2009 Department of Agricultural Biotechnology College of Agriculture Isfahan University of Technology Isfahan 84156 83111 Iran Degree M Sc Language Farsi R Zareie Assist Prof rzareie@cc iut ac ir B E Sayed Tabatabaei Assoc Prof sayedt@cc iut ac irAbstractThe maintenance of optimum conditions for growing of plants has always been in attendant by researchers In natureeach plant is attacked by the thousands of microorganisms but only small number of these microorganisms causesdisease in plants The coordination of different defensive reactions in contrast to pathogens results in stimulation ofcoping from many of defensive genes One of the important group of these genes is in charge of coding thepathogens related protein The importance of these proteins is especially because of their ability to fight on differentkinds of microorganisms Up to now 17 groups of pathogenic proteins have been recognized in monocotyledon anddicotyledon plants In 5th group of this classification are lies thaumatin like proteins Thaumatin like proteinswhich have a sequence of signal peptide in N terminal probably are acidic or neutral and are targeted to the outsideof the cell while if they have been a sequence of signal peptide in C terminal probably are alkalineand go toward vacuole For developing resistant plants against pathogenic agents thaumatin like TLP 3 gene wastransferred from one year old alfalfa plant into tobacco plant First the succession of signal peptide of the gene wasidentified by signalp software Considering the fact that this succession was in N terminal the designing of specificprimers for multiplication of the target gene was carried through PCR in 2 forms of with or without signal peptidefor targeting the protein to inside and outside of the cell respectively In the next stage we cultured of seed in MSmedium After the DNA extraction from one year old alfalfa the separation and multiplication of above mentionedgene was performed by PCR method in the presence of Pfu and Taq polymerize enzymes For the easiermaintenance and necessary access to TLP 3 gene this gene was ligated to pBluescript II SK plus pSK vector andwas transferred to E coli XL1blue E coli MC1061 Then this gene was ligated to pBI121 vector and its cloningwas done in E coli XL1blue to increase the number of pBI121 plasmids containing TLP 3 gene and to enhance thepossibility of having access to this plasmid in emergency condition In the end the mentioned plasmid wastransferred to Agrobacterium tumefaciens to transfer pBI121 plasmid containing gene to tobacco plant Afterculturing the tissues of tobacco plant in MS medium the leaves of this plant were used to cocultivating withA tumefaciens containing pBI121 Then the explant were placed in MS medium containing 100 mg l kanamaysinand 500 mg l cefatoxime were used The buds produced by callus containing the TLP 3 gene were transferred toa new medium after being separated After enough growth of tobacco plants in invitro condition they weretransferred to flower pot In this stage the DNA extraction from transgenic plants was done and then the presence ofTLP 3 gene in two forms of with and without signal peptide was proved through PCR test Also RT PCR test wasdon after the RNA extraction from the plants and mRNA synthesis was confirmed the TLP 3 gene in two forms ofwith and without signal peptide The extract of transgenic plants caused the TLP 3 protein power in preventing thegrowth of Alternaria alternata to reveal also Doing green house researches also showed the performance of thegene containing signal peptide in prevent of pathogenic behavior of Alternaria Key Words Thaumatin like gene Signal peptide Agrobacterium tumefaciens Alternaria alternata

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انتقال ژن شبه تئوماتين TLP - 3 به گياه توتون

Agrobacterium tumefaciens , َAlternaria alternata