A Preliminary Study On The Basic Characteristics Of OsMADS26 And Its Regulation Of Rice Leaf Senescence

The premature senescence of leaves seriously affects the growth and development of rice and restricts its yield and quality.Understanding the molecular mechanism of leaf senescence has important theoretical significance and application value for rice molecular breeding and yield improvement.Transcription factors play key roles in the regulation of plant senescence as a link between the nucleus and functional genes.Previous study of our lab showed that the OsMADS26 was significantly up-regulation in progeria mutant spl29.Here,we further clarified the basic characteristics of OsMADS26 and conducted a preliminary study on its involvement in the regulation of rice leaf senescence.The main results are as follows:1.The bioinformational analysis showed that the full-length CDS of OsMADS26 was669 bp and encoding 222 amino acids;OsMADS26 was a hydrophilic protein that containing MADS-box and K-box conserved domains;There was no trans-membrane region and signal peptide in OsMADS26;The secondary structure of OsMADS26 was mainly α-helix;The subcellular localization prediction of the gene was likely to be located in the nucleus.2.There are multiple cis-acting element in promoter sequences of OsMADS26 in response to various stresses and hormone signaling.We tested OsMADS26 expression at different developmental stages,stress and hormone treatment.The results showed that the expression levels of OsMADS26 was highest in leaves but lowest in stems at seedling stage,and higher in roots,leaves and leaf sheaths but lower in stems and flowers at maturity,indicating that they had specific of spatiotemporal expression;Abiotic stress,blast fungus infection and hormone treatment can induce the transcription expression of OsMADS26.For examples,the expression of OsMADS26 is up-regulated by drought,salt,Me JA and SA but down-regulated by low temperature and blast fungus;The results of self-activation function and toxicity verification showed that the OsMADS26 had no self-activation activity and the protein encoded by it had no toxicity.3.We generated OsMADS26 knockout mutants using CRISPR/Cas9 system.The osmads26 leaves retained more green coloration than WT after dark treatment;Under the MV treatment,osmads26 showed stronger ability to resist oxidative stress;The results of physiological index analysis showed that the total chlorophyll content of mutant was significantly higher than that of wild type before and after dark treatment;And the activities of CAT and SOD in osmads26 leaves were significantly than WT.4.The expression of some members of the 12-oxo-phytodienoate reductase family(OPRs)and aspartic protease family(Aps)in the mutant were down-regulated to varying degrees;OsAP25 contains multiple CARG components which combined with OsMADS;Yeast one-hybrid showed that OsMADS26 could bound to the promoters of OsAP25.5.We generated overexpression lines by Gateway technology,and phenotypic observation showed that leaf yellowing appeared at the seedling stage under natural conditions,indicating that overexpression of OsMADS26 dramatically accelerated leaf senescence.The above results indicated that OsMADS26 is a stress regulator involved in regulating the process of rice leaf senescence,and directly regulating expression of OsAP25.