Molecular Mechanism Of MicroRNA408 Adapting To Salt Stress In Maize

The area of saline alkali land in the world is about 950 million hectares,and that in China is 99.13million hectares.The increase of maize yield is restricted by large area of soil salinization.Therefore,how to make rational use of saline alkali land is an urgent problem.Although it has been reported that overexpression of miR408 enhances salt tolerance in Arabidopsis thaliana,the molecular mechanism remains to be studied.This paper uses small RNA,molecular biology,cell biology and other technical means to study the molecular mechanism of miR408 involved in maize adaptation to salt stress.The main results are as follows:1.Small RNA hybridization showed that salt stress inhibited the expression of ZmmiR408 in root and shoot.2.In order to further study the function of ZmmiR408,we constructed ZmmiR408 overexpression transgenic maize lines.According to the expression level,3#OE and 5#OE were selected for further study.The results of hydroponic culture showed that there was no significant difference in the phenotypes of WT,3#OE and 5#OE under the condition of 0 m M Na Cl treatment;when treated with150 m M Na Cl for 7 days,ZmmiR408 overexpression transgenic maize was more sensitive to salt stress,the results showed that the plant was short,old leaves are withered and the leaf area is reduced.3.Compared with the wild type,the fresh weight of the first leaf,the second leaf,the aboveground fresh weight and the chlorophyll content of the second leaf of ZmmiR408 overexpression transgenic plants were significantly decreased,and the anthocyanin content was significantly increased,which led to the decrease of photosynthetic rate,stomatal conductance and transpiration rate.4.Then,the MDA content and relative conductivity in the second leaf of maize were measured.The MDA content and relative conductivity of ZmmiR408 overexpression transgenic lines were higher under 150 m M Na Cl treatment.The Na⁺flow rate of maize mesophyll cells was further detected by Non-invasive Micro-test Technology.Under 0 m M Na Cl treatment,there was no significant difference in Na⁺flow rate among WT,3#OE and 5#OE maize mesophyll cells.Under 150 m M Na Cl treatment,the Na⁺outflow rate of ZmmiR408 overexpressing transgenic maize mesophyll cells was faster than that of wild type.5.Compared with the wild type,the content of hydrogen peroxide,superoxide anion radical and the production rate of superoxide anion radical in ZmmiR408 overexpression transgenic lines were higher when treated with 150 m M Na Cl for 7 days;we use H₂DCFDA to treat the leaves,and then observe the fluorescence to analyze the reactive oxygen species content,compared with wild-type,ZmmiR408 overexpression transgenic lines accumulated more reactive oxygen species under 150 m M Na Cl treatment,and the same results were obtained by DAB and NBT staining.6.Furthermore,the activities of antioxidant enzymes in different maize lines under salt stress were determined.Under 0 m M Na Cl treatment,the activities of antioxidant enzymes in WT,3#OE and 5#OE were not significantly different.Under 150 m M Na Cl treatment,the activities of SOD,POD and CAT in the second leaf of ZmmiR408 overexpression lines were significantly lower than those of wild type.7.The thickness of the secondary cell wall of the angle between the epidermal cells and the thickness of the secondary cell wall between the epidermal cells were observed by transmission electron microscope.Compared with the wild type,the thickness of the secondary cell wall of ZmmiR408overexpression transgenic lines was smaller under 150 m M Na Cl treatment,suggesting that the ability of ZmmiR408 overexpression transgenic maize to repair the cell wall was weaker under salt stress.8.The predicted target genes of ZmmiR408 in maize include Zm LAC9,Zm LAC18,ceruloplasmin and silk/threonine protein kinase.Real time PCR showed that only Zm LAC9 and Zm LAC18 responded to both ZmmiR408 and salt stress.Zm LAC9 and Zm LAC18 were down-regulated in ZmmiR408overexpression transgenic lines and up-regulated after 150 m M Na Cl treatment,suggesting that Zm LAC9 and Zm LAC18 may be the real target genes of ZmmiR408 in maize.Therefore,our results suggest that ZmmiR408 may be involved in the adaptation of maize to salt stress by affecting the balance of ROS and the thickness of cell wall.