The Research Of Relationship Between Transcription Factors OsMYB58/63 And OsMYB58/63-L With Leaf Senescence In Rice

Senescence of leaves shortens the functional period of rice leaves,seriously affects the growth and yield of rice,causing serious economic losses.Therefore,it is not only an important scientific problem,but also a great help to highly instructive for developing superior varieties of rice to analyze and identify the genes regulating rice premature senescence and explore the genetic basis and molecular mechanism of rice senescence.The MYB transcription factor family（TF）is one of the largest transcription factor families in plants,many studies have shown that MYB transcription factor has a wide range of biological functions,such as regulation of secondary metabolic responses,growth and development of plants,response to abiotic and biological stress,etc.However,there are few reports on MYB family genes in rice senescence.In this study,the expression patterns of Os MYB58/63 and Os MYB58/63-L in different rice tissues and their expression characteristics under abiotic stress were analyzed by q RT-PCR.The subcellular localization and transcriptional activation activity of Os MYB58/63 and Os MYB58/63-L proteins were also analyzed.Os MYB58/63 and Os MYB58/63-L knockout and overexpression mutants were created using CRISPR/Cas9 gene knockout and overexpression techniques.The premature senility phenotype and agronomic traits of the T₂ generation Os MYB58/63and Os MYB58/63-L homozygous mutants were also identified and investigated.The main research results of this paper are as follows:1.Based on bioinformatics analysis found that rice Os MYB58/63 genes located on chromosome 4 and sequence is 1952 bp and contains three exons and 2 introns,CDS sequence length is 1116 bp,and rice Os MYB58/63-L genes located on chromosome 2 and the length of 1937 bp sequence,contains two exons and introns 1,CDS sequence length is 1023 bp.The amino acid sequence analysis of the two genes showed that the two proteins had high homology（45%）,and both belonged to the R2R3 MYB transcription factor family.The promoter sequences of both genes contain a variety of cis-acting elements related to plant hormones.2.The results of subcellular localization showed that Os MYB58/63 and Os MYB58/63-L proteins were mainly distributed in the nucleus,and the transcriptional activation assay verified that the proteins encoded by the two genes had transcriptional activation activity.3.Fluorescence quantitative PCR results show that the Os MYB58/63 genes expressed quantity is highest in seedling stage of root,expressed in the young ear quantity is low,but after fertilization significantly increased the amount of seeds in its expression,Os MYB58/63-L the young ear expression quantity is high,and almost no expression in seed after fertilization.4.The wild type was subjected to high temperature,high salt,PEG drought simulation and ABA treatment.At different time after treatment,the above-ground part and the underground part were sampled.Fluorescence quantitative results showed that the expression of Os MYB58/63 gene in shoots was significantly decreased after 2 hours of treatment compared with the control.In the underground part,the expression of the gene was induced by high salinity,and the expression level increased rapidly with the increase of treatment time.After 24 hours of treatment,the expression level of the gene was more than 90 times that of the control.After ABA treatment,the expression level of the underground part of the gene increased with treatment time.The expression level of the underground part of the gene increased first and then decreased,and reached the maximum value of 35times that of the wild type after 12 hours of treatment.The expression of Os MYB58/63-L gene in shoots was strongly inhibited by high salinity,PEG and ABA treatment.The expression of Os MYB58/63-L gene decreased rapidly and remained at a low level shortly after treatment,while high temperature could promote its expression.In the underground part,the gene showed no significant change compared with the wild type in the first 12 h of PEG treatment,but its expression increased to more than 80 times that of the control after 24 h of treatment.5.Single gene knockout and overexpression mutants of Os MYB58/63 and Os MYB58/63-L T₀ generation were created using CRISPR/Cas9 gene knockout and overexpression technology.After generation isolation and molecular identification of Os MYB58/63 and Os MYB58/63-L double gene knockout mutants created,four homozygous mutants obtained in the T₂ generation were named CR-1 to 4,respectively,and these four mutants were damaged to different degrees in the conserved domain of amino acids encoded by the two genes.6.Compared with the wild type,the T₂ generation Os MYB58/63 and Os MYB58/63-L double gene knockout mutant plants showed obvious premature leaf senile.Five weeks after full heading,the flag leaves of CR-1,2 and 3 mutants were dead,and the flag leaves of CR-4 mutants also turned yellow,while the flag leaves of the wild type were still green at that time.The content of chlorophyll a and chlorophyll b in flag leaves was measured.It was found that the chlorophyll content of mutant was at least half less than that of wild type after five weeks of full heading.In the senescence experiment of dark induced leaves in vitro,the senescence rate of mutant was also faster than that of wild type.7.The yield characters of wild-type and premature senescence mutants were investigated.It was found that there were no significant differences in plant height,number of primary and secondary branches,spike length and total grains per panicle between wild-type and premature senescence mutants,but the seed setting rate,1000grain weight and yield per plant of mutant were significantly lower than those of wild-type（control）.Compared with the wild type,the seed setting rate of CR-1,CR-2 and CR-3 decreased by about 40%,the 1000 grain weight decreased by about7%,and the yield per plant decreased by about 60%.The senescence rate of CR-4mutant was slower than that of other mutants,but faster than that of wild type.Compared with the control（wild type）,the seed setting rate of CR-4 mutant decreased by about 20%,the 1000 grain weight had no significant difference,and the yield per plant decreased by about 40%.