Cloning And Functional Analysis Of Early Senescence Leaf Gene ESL12 In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)belongs to Gramineae,which is not only a monocotyledonous model plant but also an important food crop.Under the background of increasing population and environmental pollution,the research and development of high-yield and high-quality rice plays an irreplaceable role in human survival.The accumulation of carbohydrates in rice mainly comes from the photosynthesis of leaves,and the early aging of rice leaves will make the chlorophyll of plants degrade in advance,which may be accompanied by the change of leaf color,affect the photosynthesis of leaves,and then affect the normal accumulation of carbohydrates.Therefore,it is of great significance to explore the function and molecular mechanism of rice leaf premature senescence gene for improving rice yield and quality in China.In this laboratory,EMS(ethyl methane sulfonate)was used to mutate ‘Xinong1B’,a three line maintainer of indica rice,and a starch accumulating early senescence mutant esl12 was selected from the progeny of its stable genetic mutant.Through 70 mutants,ESL12 gene was located on the third chromosomal;in this study,the mutant was further refined to obtain ESL12 candidate genes,and functional complementation verification was performed.The ESL12 protein structure and function,gene expression specificity,transcriptome and uric acid content was analyzed,and the following conclusions were drawn:1 Gene location and cloning of ESL12Compared with the wild type,the rice esl12 mutant showed the early senescence of leaf tip from seedling stage,and then gradually extended to the middle of the leaf,and the leaf base was light green,which lasted to the maturity stage.In addition,a large amount of starch was accumulated in the leaves of the esl12 mutant.On the basis of gene mapping carried out in the laboratory,the content of uric acid in the leaves of wild-type and esl12 mutants was determined.The results showed that the content of uric acid in the leaves of wild-type and esl12 mutants was significantly lower than that of wild-type;Further development of molecular markers to locate the gene in the range of 252.7 kb between BB-1 and BB2-1,sequencing the candidate genes in the range,found that the 20 th base of the fourth exon of Os03g0429800 in the mutant esl12 was replaced from A to T,which made the corresponding amino acid from histidine to leucine,and Os03g0429800 was initially identified as the candidate gene of esl12.To verify whether the premature aging phenotype of esl12 mutant was caused by the mutation of Os03g0429800 gene,the 4110 bp CDS sequence of Os03g0429800 gene wild type was cloned,an overexpression vector was constructed and the callus of esl12 mutant was transformed,and the leaves of the positive transformed plants all returned to green.The results show that the Os03g0429800 gene is the ESL12 gene.2 The protein analysis of ESL2Conservation analysis of protein amino acid sequence and analysis of protein structure characteristics show that ESL12 gene encodes a conserved xanthine dehydrogenase(XDH,Xanthine dehydrogenase),which is conservative in evolution,and is distributed in animals and plants.Leaf plants such as sorghum,maize,millet,and Erwinia brevifolia have similar XDH kinship.The ESL12 protein contains two amino-terminal ferredoxin domains(Ferredoxins),a FAD binding domain,and a molybdenum cofactor binding(Molybdenum cofactor binding)domain.The full-length CDS of wild-type ESL12 gene was cloned,and a 35S::ESL12-GFP subcellular vector was constructed.The rice protoplast was transformed by PEG,and the fluorescence signal was observed by confocal microscope.The results showed that the ESL12 protein was located in the cytoplasm.3 Expression pattern analysis of ESL12 geneQRT-PCR was used to analyze the expression patterns of different tissues of wild type and mutant plants at tillering stage and young spikes at heading stage.The results showed that ESL12 was expressed in roots,stems,young leaves,old leaves,sheaths and young ears of wild type and mutant,and the expression level in young leaves and old leaves was higher than that in other tissues;in addition,in the same tissue,the expression level of ESL12 in mutant was generally higher than that in wild type.4 Expression and transcriptome analysis of photosynthetic pigment metabolism,chloroplast development and photosynthesis related genesThe expressions of genes related to chlorophyll and carotenoid metabolism and genes related to chloroplast development and photosynthesis in the tillering stage of wild-type and esl12 mutant leaves were analyzed by q RT-PCR.The results showed that in the esl12 mutant,genes for chlorophyll synthesis pathway-related enzymes including CAO1,CHL1,DVR,and HEMA1 were significantly down-regulated;while genes for carotenoid synthesis pathway-related enzymes had a slight increase in PSY3 transcription levels,other genes such as PDS,PORA,PSY1 and PSY2 were down-regulated to varying degrees;In addition,the chloroplast development and transcription levels of photosynthesis-related genes in the mutants were down-regulated to varying degrees.Transcription analysis of wild-type and mutants showed that the mutant purine metabolism pathway was severely affected;pigment metabolism,chloroplast development and photosynthesis in the mutant were inhibited,consistent with q RT-PCR results;ESL12 gene mutations affected genes related to reactive oxygen species(ROS).