Cloning And Analysis Of Expression Characteristics Of Zmstomagen Gene In Maize

Plant stomata is the most important aisle for gas changing with the environment. The capacity and efficiency of plant photosynthesis are regulated by stomatal aperture and density on leaf epidermis. So that the researchers have been focused on stomatal development and patterning in plant developmental biology. In the stomatal developmental pathway of the model plant Arabidopsis thaliana, by now, Stomagen/STOMAGEN is the only secretory peptide identified that plays a positive role in stomatal development. It is reported that, promoting stomatal development by increasing the level of STOMAGEN in Arabidopsis could result in30%increase of photosynthesis efficiency, However, in monocot crops, no research about this has been reported.Maize is a monocot model plant, as well as one of three most important crops in the world. Functional study on maize’STOMAGEN’will shed more insight on mechanism underlying monocot stomatal development and patterning. Furthermore, the scientific theory for cultivating high photosysthesis efficiency monocot crops will be built up undoubtedly on the research results. In this study, gene ZmSTOMAGEN from Zea mays Chang7-2was cloned and characterized based on the theories of reverse genetics and qPCR. The results are shown as follows:1. ZmSTOMAGEN was cloned from Chang7-2by sequence analysis and PCR technique. The cloning sequence of this gene is identical to the coding domain sequence of NM₀01149748.1in Zea mays B73in NCBI. The bioinformatic analysis indicated that, there are123amino acid residues in ZmSTOMAGEN, the structure of ZmSTOMAGEN is similar to STOMAGEN, which contains a signal peptide in its N-terminus, a mature peptide （zmStomagen, containing6conserved cysteins residues） in its C-terminus, a casein kinase Ⅱ phosphorylation site and a protein kinase C phosphorylation site, a loop, two beta strands and an alpha helix comprise the active three-dimensional structure depending on three intramolecular disulfide bonds. The similarities of the whole peptide and the mature peptide between ZmSTOMAGEN and STOMAGEN are48%and82.22%, respectively.2. Various cis-regulatory elements such as’mesophyll expression module1’ elements, shoot expression regulatory elements, light response elements and water response elements were found in promoter upstrem region of ZmSTOMAGEN, it suggested that, the expression of ZmSTOMAGEN is tissue-specific, light-and drought-induced.3. ZmSTOMAGEN mainly expressed in tissues where stomata distribute on and cells are growing, such as the leaf sheaths, immature leaves, coleoptiles and mature roots. The highest and the lowest expression level was detected in leaf sheaths and mature roots, respectively. But there was not any expression detected in mature leaves and immature roots. The tissue-specific expression characteristic of ZmSTOMAGEN described above appeared similar to STOMAGEN.4. The expression of ZmSTOMAGEN and the alteration of stomatal index on abaxial epidermis of new leaves showed inducible under different light intensities:under5000lx,600lx and1801x, the expressions of ZmSTOMAGEN were1,0.667and0.131, respectively; the stomatal indexes were20.15±1.10%,17.22±1.39%and15.38±1.87%, respectively. As the light intensity turning weaker, the change trends of ZmSTOMAGEN expressing and stomatal index appeared similar to each other.5. The expression of ZmSTOMAGEN and the alterations of stomatal index and morphology on abaxial epidermis of new leaves showed inducible under different relative water content in soil. As the drought turning heavier, the change trends of ZmSTOMAGEN expressing and stomatal index appeared similar to each other, but the change trends of ZmSTOMAGEN expressing and stomatal length and width appeared negative to each other: when the relative water content were80%,50%and30%, the relative expression of ZmSTOMAGEN were1,1.14and1.73, respectively; the stomatal indexes were18.74±1.42%,20.71±1.06%and21.93±1.29%, respectively; the stomatal lengths were478.14±9.17μm,444.92±11.57μm and423.30±9.31μm, respectively; and the stomatal widths were25.04±2.20μm,23.34±2.65μm and22.02±2.31pm, respectively.6. With the plant binary vector pGSA1252as backbone, an interference vector （pGSA1252-RNAi） and an overexpression vector （pGSA1252-OE） were successfully constructed. These two vectors were introduced into Agrobacterium tumefaciens.In summery, the structure of protein encoded by ZmSTOMAGEN, the promoter cis-regularity elements and the tissue specificity of ZmSTOMAGEN gene in Chang7-2were compared with STOMAGEN from Arabidopsis. Besides, the light-and drought-induced change trends of the ZmSTOMAGEN expression and the stomatal index appeared the same with each other. All the data suggest that, ZmSTOMAGEN gene cloned from maize Chang7-2is likely the key player in maize stomatal development. The RNAi and overexpression vectors constructed successfully will aid the next functional study of ZmSTOMAGEN gene.