Molecular Mechanism Of French Bean Gene PvSR2Expression Regulated By Its Intronic Promoter

Intron is one of the important characters of eukaryotic genes. The expression level and the diversity of gene can be regulated by intron and its splicing process from pre-mRNA. In animals usage of alternative intronic promoter (AIP) is a critical method to increase the variety of gene’s expression. So far only a few AIP have been reported in plants, including the French bean heavy metal response gene PvSR2elucidated in our previous research. PvSR2gene has two promoters:the upstream one (named P1) responding to heavy metals (Cd, Cu, Hg, etc) and the intronic one (named P2). PvSR2produces a new transcript (named S-PvSR2) via P2and S-PvSR2encodes a novel transcription factor NP2. Overexpression of NP2in French bean leaves increased the accumulation of PvSR2transcript, suggesting that NP2positively regulates PvSR2. Based on these achivements we studied the impact of Cd on P2and NP2and tried to shed light on the molecular mechanism of PvSR2expression regulated by NP2.1. GUS reporter driven by P2is mainly expressed in stems and leaves in tobacco and P2respond to Cd inductionTransient expression analysis showed that GUS reporter gene driven by P2was mainly expressed in stems and leaves in tobacco. qRT-PCR analysis indicated GUS expression was increased significantly under Cd stress in two P2:GUS stable transgenic tobacco lines. P2is able to respond to heavy metal stress.2. NP2is a Cd-responding transcription factorSince P2is induced by Cd it is assumed NP2driven by P2can respond to Cd as well. We confirmed this hypothesis on mRNA and protein level. We used qRT-PCR to detect the change of P2transcript S-PvSR2in French bean under Cd treatment (100μM CdCl224h) and found that its expression level was up-regulated14-folds compared to the control. The detection of NP2by Western blot revealed NP2accumulation in French bean leaves was increased significantly up to1.6times. These demonstrate that NP2is a transcription factor responding to Cd stress. 3. NP2binds directly to the heavy metal response segment on PvSR2gene promoter to regulate PvSR2expressionFurther experiments were carried on to study if NP2binds to PvSR2promoter directly. We proved that NP2bound to the specific76-bp segment on PvSR2promoter (-222/-146) by conducting the yeast one-hybrid experiment (Y1H), chromatin immunoprecipitation assay (ChIP) and reporter gene assay.Former transient expression experiment in protoplasts showed there is one metal response element (MRE) in that76-bp region. GUS expression was up-regulated driven by76-bp segment of PvSR2minimal promoter in tobacco under Cd treatment, suggesting76-bp is capable of responding to heavy metal. It is inferred that NP2binds to MRE in this region. Further electrophoretic mobility shift assay (EMSA) analysis confirmed the fact that NP2binds to the MRE within76-bp in vitro.In all we proposed a novel model of plant gene regulation using AIP based on our study of PvSR2intronic promoter P2, the transcript S-PvSR2and transcription factor NP2:French bean gene PvSR2produces a Cd-responding transcription factor NP2through intronic promoter P2; NP2binds to the MRE on promoter P1to enhance the expression of PvSR2to form a self-regulation loop.