Cloning Of Maize Myo-INOSITOL-1-PHOSPHATE SYNTHASE Gene (ZmMIPS) And Functional Characterization Of Its Drought Tolerance

Maize(Zea may L.)is one of the important crops in the word.The production of maize crop is seriously affected by drought stress all over the world.Previous studies have shown that over-expression of L-myo-inositol-1-phosphate synthase gene(MIPS)enhances salt-,cold-and oxidation tolerance of plants.MIPS is the rate-limiting enzyme of inositol biosynthesis,it catalyzes G-6-P to generate L-myo-inositol-1-phosphate.The IMP(myo-inositol monophosphatase)then catalyzes L-myo-inositol-1-phosphate to generate myo-inositol.Myo-inositol and its derivatives play important roles in plant growth and development.Using homology cloning methods,two members of maize ZmMIPS gene,encoding L-myo-inositol-1-phosphate synthase,were cloned and characterized.The main research results are shown as following:1.Two members of ZmMIPS,ZmMIPS1(GRMZM2G155242)and ZmMIPS2(GRMZM2G004528)were found in the maize genome.Both ZmMIPS1 and ZmMIPS2 are able to synthesize L-myo-inositol-1-phosphate in vitro.2.Kinetic analysis showed that ZmMIPS2 has higher Km for G-6-P than that of ZmMIPS1.The dependence on NAD~+content between ZmMIPS1 and ZmMIPS2 is different.3.ZmMIPS1 and ZmMIPS2 are expressed in most tissues.ZmMIPS2 expresses at higher level in leaves and embryos compare with that of other tissues.Both genes express at higher levels in the early and middle stages of embryo development.The expression of both ZmMIPS is induced in maize seedlings by heat,osmotic stress,MV and ABA.4.The dual luciferase expression vector,in which Rluc expression was controlled by the ZmMIPS promoter and Fluc expression was driven by the 2×35S promoter,was constructed.The dual luciferase expression vector was co-transformed into maize protoplasts with either ZmDREB2A or GFP expression vectors.The Rluc expression was significantly increased by over-expression of ZmDREB2A compared with that of GFP over-expression.This suggests that ZmDREB2A up-regulate the expression of ZmMIPS1 and ZmMIPS2.5.Over-expression of ZmMIPS in Arabidopsis increases myo-inositol content in mature leaves,and enhances plant drought tolerance.In summary,I have characterized two maize ZmMIPS genes.Both ZmMIPS1 and ZmMIPS2 are induced by multiple abiotic stresses.ZmDREB2A up-regulates the expression of ZmMIPS1/2.Over-expression of ZmMIPS1/2 in Arabidopsis enhanced plant drought torelance.