Cloning of myc tagged rolA gene in Agrohacterium-mediated plant transformation vector

Abstract

It is a proven fact that secondary metabolites like alka loids. stero ids etc. are cruc ial for lead identili cation. optim izati on. dru g discove ry and deve lopme nt. Because most of the small molec ule drugs are cOlllpl ex orga ni c Ill olec ular scaffolds that arc difficu lt to synthesise in vitro. current ly plants are considered th e best source fo r their commerc ial supp li es. Howeve r. sma ll amo unt o r these metabo lites in ce lls is bottleneck in their large sca le extract ion. due to the hi gh cost in terms of time and money. So understanding the mol ecular biology of plants as we ll as va ri ous genes that boost these metabolic flu xes is a good strategy in this regard. The rol genes derived from Agrobacterium rhizogene are widely used in this regard but the exact mechanism of how rot genes enhance secondary metabol ites is not known. Hence rol genes functi onal proteomi c detailed understandi ng is essential in their rational manipulation. This study was designed to shed light on this aspect by constructing epitope tagged rotA gene by adaptor pe R (N-termin a l III.1'C taggin g by usin g ove rl apping se t of primers) 81 Y end or rotA gene I'o ll o\\ ed by its forced c lonin g into pEarlyGate203 expression vector for !lgro/JuCl l.'rilllll lillll(/cic iens mediated transformati on. Thus resulting recombinant plasmi d pMBQAU 100 I harbours CaMV 35S promoter, Kan bacterial selectab le marker gene and plants selectable marker gene BASTAr. Initially competent ce lls of E. Coli DH5a. vve re tran sformed with this construct by e lectroporation and co lonies were successfully screened by PCR as we ll as by restriction mapp ing. Later, pMBQAU 100 I was electroporated into competent ce lls of disarmed Agrobacteriul11 lumifaciens GV3 JOJ followed by screen ing and validation of cloning by using gene spec ific PCR primers fo r both NPTll (Kan) and rolA ge ne as we ll as fina l contirmation by sequenc in g of myc-rotA gene fragme nt. Prospectively, this transfo rmed Agrohaclel"llllllllll7l1Iuciel1 C;V3JO Jstra in can be used in future for fun cti onal proteoillics studi es 01' mt,4 gene incl uding subce llular immunolocali zation and affinity purification.