Identification And Characterzation Of ERF And RAV Transcription Factors From Galega Orientalis

Galega. orientalis L., a species in leguminous Galega is a kind of good fodder pasture. Galega. orientalis L. has much value in use, such as high productivity, chronic utilization, resistibility, good palatability, etc. Because of these advantages, it not only improve cow's milkability and milk fat percentage,but also establish and improve artifiicial pasture. It has a best foreground in"grain for green, return grazing land to grassland, leyfarming". Therefore, it is valuable to deeply study characters and functions of Galega. orientalis L. to develop economic and ecological significance. In order to understand molecular mechanism of Galega. orientalis L. to various abiotic stresses, in this paper,two DREB-like genes were isolated by RACE methods from Galega. orientalis L.,and their sequence characters,expression patterns and functions were deeply analyzed to understand clearly molecular mechanism of plant response to various abiotic stresses. The main results are follows:1. Isolated a ERF transcription factor of Galega. orientalis L.in cool condition. Analyzed the gene's some characters and functions.(1) A cDNA clone encoding ERF-binding protein, designated GoDREB. The gene's code area has 1095bp in length which encode 364 Amino Acids. Analyze sequense show: GoDREB gene has an introne of 1332 bp which is located between 255bp to 266bp in open read frame. GoDREB protein has three characteristic domains of the transcription factor, namely an AP2/EREBP domain,a nuclear localization signal and an acidic activation domain.The deduced amino acid sequences of GoDREB showed significant sequence similarity with those of other known ERFs ,expecially in the conserved AP2/EREBP DNA-binding domain. GoDREB has typical characters of ERFs transcription factors. Consequently, GoDREB protein is a new member of ERFs transcription factors, which is belong to classIV subfamily. (2) Analyzed to understand clearly expression patterns of Galega.orientalis L. response to various abiotic stresses by real time PCR. Consequenses show: expression of GoDREB gene not only be induced by low temperature, PEG and salty,but also induced by exogenous hormone MeJA and SA. Thereinto, The cool induction is obviousely earlier than drought and salinity. No significant changes in the GoDREB expression were observed under ABA.Thereafter, GoDREB resist various abiotic stresses by the accommodation of SA/JA, which is not dependent on ABA. Analyze specificity expression of GoDREB gene in different organization by RT-PCR. Result show: it has some discrepancies in three organizations. It was expressed strongly in leaves, and faintly in roots. Thus it can be seen, GoDREB gene belong to constitutive expression genes.(3) Analyzed result show: GoDREB protein was located in the cell nucleus. Fusion protein pCAMBIA1302-GoDREB-GFP was mainly distributed in the cell nucleus. But pCAMBIA1302 were distributed in the whole cell. It is demonstrated that GoDREB protein has the trait of nuclear localization.(4) We transformed GoDREB gene into tobacco with Agrobacterium tumfaciens containing an expression vector by Agrobacterium infestation. Detect T0 generation of transgenic tobacco by PCR.The results showed: GoDREB gene has been transformed into tobacco and expressed in it.2,Isolated a RAV transcription factor of Galega. orientalis L. in cool condition and analyzed the gene's some characters and functions.(1) A cDNA clone encoding binding protein, designated GoRAV. The gene's code area has 1164bp in length which encode 387 Amino Acids. Analyze sequense show: GoRAV gene did not have introne. GoRAV protein is a new member of RAVs transcription factors. The amino acid sequences of GoRAV is more similar with GmRAV's. The deduced amino acid sequences of GoRAV showed significant sequence similarity with those of other known dicotyledon.(2) Analyzed to understand clearly expression patterns of Galega.orientalis L. response to various abiotic stresses by real time PCR. Consequenses show: expression of GoRAV gene was induced by low temperature, PEG, salty and exogenous hormone MeJA and SA. Thereafter, GoRAV resist various abiotic stresses by the accommodation of ABA/MeJA,. Analyze specificity expression of GoRAV gene in different organization by RT-PCR. Result show: it was expressed strongly in leaves and stem.(3) Analyzed result show: GoRAV protein was located in the cell nucleus. Fusion protein pCAMBIA1302- GoRAV -GFP were mainly distributed in the cell nucleus. But pCAMBIA1302 were distributed in the whole cell. It is demonstrated that GoRAV protein has the trait of nuclear localization.(4) We transformed GoRAV gene into tobacco with Agrobacterium tumfaciens containing an expression vector by Agrobacterium infestation. Detect T0 generation of transgenic tobacco by PCR.The results showed: GoRAV gene has been transformed into tobacco and expressed in it.