Kloning Fragmen Gen cryIA Penyandi Domain Toksin Menggunakan Vektor pGEM-T

Abstract

Cry is ollc of tllc n~ost witlcly used gcncs in plant gcnctic engincclilll: for pcst resistance. Cloning of this resistancebearing ):en.: from Bocillrrs ~huringierrsis has been cortduc{ed by scrcc~lit~thge gcnon~icl ibrary. Thc disadvantages of such a cloning approach arc thc cornplcxily of the proccdurc and the scrccllcd clor~cs are often not intact. They contain useless flnnking regions or a part of open reading fran~e(O RI.') is truncated. A simpler and direct cloning proccdurc is amplification of the gcne using specific primer, and ligation of the amplificntion product into 7-vector This rcscarch aims to clcne crylA gene with (ha of direct proccdurc. The I'CI< producl of crylA gene fragment was ligated with pGEM-T vector, followed by transformation into Esclrerichia coli DH5a and JM 109. Transformed cells were grown OII selection media containing 50 mgll ampicillin. 40 mgll X-Gal, and 0.1 mM II'TG. Analysis o f recombinant plasmid (5 kb) from white colol~icsw ere performed on agarosc gel. Results shokcd that of (IIC four crylA fragments used, those from B. lhuringien.rls subsp. kursfoki of the FL and CD isolatcs produced 2 and 3 cloncs and were identilied to carry pGEM/ cryIA(c) rccombinants.