| Early leaf senescence not only results in the decrease of individual photosynthetic efficiency,reduced photosynthetic product,and the recycle of the nutrient elements from vegetative organs(mainly leaf)to reproductive organs and storage organs(mainly seed)are also affected.It seriously affects the corn yield development.In the paper,two leaf senescence extreme phenotype inbred lines(ELS-1 and Yu87-1)were choosed and analyed the leaf senescence transcript,the dynamic changes of the expression of mi RNAs,identified the functions of differentially expressed genes and mi RNAs in different development period,and explored the regulation mechanism and the molecular mechanism in the process of leaf senescence.Besides the simplified genome sequencing technology was employed to identify the senescence associated genes,and the candidated genes were predicted.The main results are as follows:1.After pollination,the leaf senescence are first started from the bottom of the leaves,and the yellowing is happened from the tip of the leaf,and extend to the interior of the leaf;After that,the leaf senescence of the whole plant is extent from bottom and top leaves to middle part of the leaves gradually,and the ear leaf is begun to senescence at last.It is well known that the degradation of chlorophyll content is the external indication for leaf senescence.At the latest stage of the leaf senescence,the content of chlorophyll a,chlorophyll b and total chlorophyll content were decline in both of the two inbried lines,but the pattern was different from each other.Compared with Yu87-1,the early leaf senescence line,ELS-1,has a faster and earlier chlorophyll content decline at the late stage of leaf senescence.Besides,at the 30 DAP,the content of chlorophyll was quite low.As for Yu87-1,the decreasing range of chlorophyll was a little slow during 10-30 DAP,and the decline of the chlorophyll b begun at 25 DAP,later than that of ELS-1.2? Using large-scale high-throughput sequencing platform Illumina2500,four libraries were constructed in leaf senescence process of the two inbred lines for transcriptome sequencing.As the aggravation of the ageing,1351 different expression genes were found on ELS-1 with 881 up-regulation genes(65%)and 470 down-regulation genes(35%)genes.The most enrichment pathway of different expression genes concented in plant hormone signaling interactions,photosynthesis,carbon fixation,photosynthesis,nitrogen metabolism,arginine and proline metabolism as well as starch and sucrose metabolism pathways.3?Based on the Illumina technology,mi RNA-seq of two inbred lines we carried out at 20 DAP and 30 DAP.215,506 clean reads were obtained in total.After data cntrol,80 differental expression mi RNAs were identified in ELS-1.These differental expression mi RNAs were belong to 26 mi RNA families.Analysis the expression pattern of these difference mi RNAs,37 mi RNAs had same expression patterns totally.and Among them,15 of these mi RNAs were up-regulated at 20-30 DAP,and 22 of them were down-regulated,while the other 44 had differential expression pattern between the two inbred lines.According to the sequence and second structure characteristic of mi RNA,164 new mi RNAs were obtained.,The target gene of mi RNA were identified through the degrome sequence,and 339 target genes of 48 known mi RNAs were charactered.Through the analysis target genes of different expression mi RNAs,zma-mi R156,zma-mi R159,zma-mi R167,zma-mi R171,zma-mi R172,zma-mi R395,zma-mi R399,zma-mi R408 and zma-mi R529 were found involved in leaf senescence regulated.The main pathway those target genes participated were leaf development,chlorophyll degradation,transcriptional regulation,signal transduction and stress response.These results indicated that mi RNA play important role in leaf senescence development regulation.4?Using SLAF-seq,associated analysis were carried out on maize leaf stay-green BC1 genetic segregation population(male and famale parents,10 plants for each two phenotype).Using maize B73 as reference genome,we conducted electronic enzyme,and Hae III+Rsa I were selected for digestion.The fragment length of the restriction digestion is 414-444 bp,and the cleavage efficiency is 89.27%.Finally,19.73 M reads were obtained of the four libraries.Through the biology information analysis,165,127 SLAF markers were charactered.Among these markers 36,673 SLAF markers,22.21% of total SLAF markers,were polymorphism markers.Through ED analysis,2 hot-regions were located on chromosome 3.Function annotation and biology pathway enrichment analysis were carried out by KEGG,Combain with the phenotype of the BC1 genetic segregation population,arginine and proline metabolic pathway might participted the leaf senescence regulation.5? Based on the data different expression profile of transcriptome,mi RNA and associated analysis of SLAF-seq,conjoint analysis was carried out through bioinformatics methods.On the proline metabolic pathway acquired by conjoint analysis,mi RNAs and its target genes constructed a complex interaction network through different funtional gene cluster node.Among them,mi R399,mi R529 and mi R159 were identified to connect with the candicated gene GRMZM2G068665,and participated on the regulation pathway during leaf senescence through adjusted the biosynthesis of proline. |
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