Digging And Functional Identification Of Key Genes In Rice Crown Root Development Based On Microdissection And Transcriptome Sequencing

Crown root is the main component of rice root system,and its root growth and development are closely related to rice yield and environmental adaptability.In the early stage,our research team used laser microdissection technology to sample and perform transcriptome analysis on the four key stages in different root primordium developmental stages(S0-S3).In order to analyze the genes that regulate the development of different stages in crown root primordium,this study focused on the expression of genes related to hormones and transcription factors at each stage,and identified some differentially expressed genes in adjacent stages,and elucidate the functions of these genes in rice crown root development through genetic methods.The main research results are as follows:1.Enrichment analysis of differentially expressed genes revealed that down-regulated genes during the S1 stage were enriched in genes related to cytokinin biosynthesis,while up-regulated genes during the S1 stage were enriched in genes related to cytokinin degradation;The auxin related genes PIN,ARF and IAA were specifically overexpressed at S0 and S1 stages;The up-regulated genes during S3 stage are enriched in genes related to gibberellin biosynthesis;The ethylene signaling genes are specifically overexpressed during the S3 stage,indicating that the above-mentioned hormone related genes may be involved in the development of rice crown root.2.Analysis of up-regulated transcription factors in adjacent periods revealed that 25 transcription factor families,including SPL,LBD,PLT,WOX,b ZIP,and MYB were up-regulated.Among them,144,108,and 120 transcription factor genes were up-regulated in S1,S2,and S3 periods,respectively.Then OsbZIP08 and Os MYB110 were selected from the up-regulated transcription factors in the S1 stage,and OsbZIP65 was selected from the up-regulated transcription factors in the S2 stage.In addition,in previous work,it was found that the gene OsCBP(Calmodulin-Binding Protein)was only highly expressed in the S1 stage.We used these four genes as candidate genes to explore their functions in crown root development.3.Constructing and examineing the phenotypes of each mutant revealed that,compared with the wild type,the number of crown root and the length of primary root in bzip65,cbp and bzip08 after 7 days of germination were decreased.There was no significant difference in the number of crown root and the length of primary root between myb110 and the wild type.OsbZIP65 and OsCBP were selected for subsequent research.The results of in situ hybridization experiments showed that OsbZIP65 was highly expressed in S0 stage and OsCBP was highly expressed in S1 stage.The paraffin section results showed that the initiation and growth of crown root primordia of bzip65 and cbp were affected.The semi thin section results showed that the number of cells in the root tip meristem of bzip65 and cbp were decreased.The above results indicate that OsbZIP65 and OsCBP are necessary for the initiation and growth of rice crown root.In summary,this study found that genes related to auxin,cytokinin,gibberellin,ethylene,and transcription factors are specifically overexpressed at different stages in rice crown root development.OsbZIP65 and OsCBP participate in regulating the development of rice crown root.This study analyzed the molecular regulatory network of rice root development.