Preliminary Characterization Of Arabidopsis Bidirectional Promoter K9I9 In Maize

In plant gene function research and transgenic plant breeding,there requires constructing more than one gene into a single vector that each functional gene usually has its own independent set of regulatory elements.This increases the length of the T-DNA region leading to either complexity in vector construction or the difficulty in genetic transformation.In practice,thus,construction of a smallest plant expression vector that harbors multiple genes has become an important technical aspect in transgenic plant research and application.In fact,the length of a given functional gene is immutable,what can be changed is the regulatory element section within the multiple cloning sites.Bidirectional promoter is a regulatory sequence present in many natural organisms.It promotes the expression of a given gene in both orientations of its sequence.Discovery of bidirectional promotors provide new insight in efficiently vector construction of functional gene.In literature,many efforts have been made in the functionality validation of bidirectional promoters for their both sequence orientations.However,there has hardly been report describing the promoting efficiency of bidirectional promotors.In this study,a bidirectional promoter K9I9 from Arabidopsis was adopted to construct plant expression vectors with gus as reporter gene,so as to determine the functionality and the promoting efficiency of K9I9 in transgenic maize.Pilot experiment showed that gus transcripts may be detected in the immature embryo of transgenic maize,suggesting that K9I9 functions well in transgenic maize.Further,a Lepidoptera-resistance gene cryNGc and a glyphosate-resistance gene epsps were co-constructed at different orientations of K9I9,with2×35S::bar as screening marker gene,forming a bidirectional expression vector cryNGC::K9I9::epsps.Genetic transformation of this vector gave rise to at T0 generation 25 transgenic maize of 5 events,with which the bidirectional functionality of K9I9 in transgenic maize was evaluated.RT-PCR results showed that the exogenous genes on both sides of K9I9 expressed in T0 and T1 transgenic maize;real time-PCR showed that the promoter efficiency of K9I9 was ca.13.1%-70.0% of 2x35S::bar and,K9I9 exhibited a promoting efficiency to the functional genes at both sides as cryNGC 3.25×106 copies/?l and epsps 17.4×106 copies/?l respectively.Bioinformatics analysis indicated that differences in quantity and nature of conventional regulatory elements such as TATA-box etc.are present in the forward and reverse orientations of K9I9.In summary,Arabidopsis bidirectional promoter K9I9 functions in both orientations with divergent efficiency in the transgenic maize.It is at moment unclear the reason affecting the efficiency of the bidirectional promoter.The current results suggested that the Arabidopsis K9I9 promoter may be used in maize breeding.