Cloning Of Key Gene SiNCED3 In ABA Pathway Of Foxtail Millet And Its Response To Stress

As a regulator of plants,ABA can induce plants to produce resistance to adverse environment（drought resistance,cold resistance,disease resistance,salt tolerance,etc）,and it is the"stress inducing factor"of plants.The application of exogenous ABA can effectively alleviate the damage caused by drought stress to plants,9-cis-epoxy carotenoid dioxygenase（NCED）is one of the key enzymes in the biosynthetic pathway of ABA.At present,a large number of studies have proved that nced gene regulates crop growth and development,but there are few reports in foxtail millet.Foxtail millet is a C₄cereal model plant with great development potential.Studying the function of Si NCED3 in millet provides an important theoretical basis for studying the stress resistance mechanism of millet and cultivating new varieties of millet with stress resistance.In this study,through the application of exogenous ABA,the effect of ABA on different drought-tolerant millet was studied.Secondly,the drought-tolerant millet variety Jigu 22 was used as the material to clone Si NCED3,and its function was preliminarily studied.The results are as follows:1.The application of exogenous ABA can significantly reduce the accumulation of malondialdehyde（MDA）in millet seedlings under drought stress,promote the significant increase in the activities of antioxidant system-related enzymes superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）,ascorbic acid peroxidase（APX）,as well as the osmotic adjustment substances proline,soluble sugar and a large amount of soluble protein was accumulated to alleviate the damage of millet seedlings caused by drought stress.At the same time,it was found that the application of exogenous ABA had a more obvious effect on the drought-tolerant variety‘Jigu 22’under drought stress.2.Using Jigu 22 as material,Si NCED3 gene of millet was cloned by RT-PCR（NCBI accession number is OM674734）.The total length of CDs gene is 1836 bp.NCBI-Blast shows that the nucleotide similarity with rice Os NCED3（XM＿015776052.1）is as high as 89.63%.Bioinformatics analysis of Si NCED3 shows that Si NCED3 encodes 611 a mino acids,with a relative molecular weight of 66.36k D and an isoelectric point of 6.15.Through the instability coefficient,it is speculated that Si NCED3 protein is a stable protein without signal peptide,which contains the typical domain RPE65 of the NCED gene family,and the coils and extended chains constitute the secondary structure of Si NCED3,Subcellular localization predicted that it was located in chloroplast.Phylogenetic analysis showed that foxtail millet Si NCED3 had the closest phylogenetic relationship with maize（Zea mays）Zm NCED4,which was also a monocotyledon C₄plant,followed by（Setaria viridis）Sv NCED1,and the farthest phylogenetic relationship was dicotyledon tobacco（Nicotiana tabacum）Nt NCED1 ntnced1.The analysis of cis acting elements of promoter showed that it was regulated by hormones such as light,abscisic acid and auxin.3.The q RT-PCR analysis showed that Si NCED3 was expressed in different tissues of root,stem,leaf,leaf sheath and spike,and the expression in root was the highest.Through different stress treatments,it was found that drought,low temperature and salt stress could induce the expression of Si NCED3 in Millet Seedlings to varying degrees.4.The p C1300s-Si NCED3 expression vector was constructed and transformed into Arabidopsis by inflorescence soaking.Through different stress treatments,it was found that overexpression of Si NCED3made Arabidopsis seeds more sensitive to ABA at the early stage of germination,enhanced the tolerance of Arabidopsis seedlings to ABA.Under drought and salt stress,overexpression of Si NCED3 transgenic Arabidopsis can promote the synthesis of chlorophyll,POD,SOD,CAT,and proline in leaves,while reducing the accumulation of MDA.The above results indicated that overexpression of Si NCED3 improved the drought and salt tolerance of Arabidopsis.