Dissection Of Coding Genes And Long Non-coding RNA Related To High Temperature Stress At The Seedling Stage In Rice

Rice is the staple food of nearly one-third of the world's population and is an important food crop in China.However,the global warming,especially the extreme high temperature weather,poses a serious challenge to the world's rice production.Therefore,clarifying the molecular mechanism underlying rice heat tolerance,identifying related gene resources?including coding and non-coding genes?and breeding heat-resistant rice varieties will be vital to cope with this challenge.In this study,two rice cultivars with significant differences in heat resistance: ZS97B?heat resistant?and SYD2?heat sensitive?were treated with high temperature stress at the seedling stage,and three replicates of each cultivar were sampled at 0,2 and 6 days after treatment.Then,all samples were performed for strand-specific transcriptome sequencing?ssRNA-seq?;the gene expression profiles of the samples were compared and analyzed,and the differentially expressed genes?DEGs?were screened for functional analysis;the heat responsive core genes were mined by constructing a regulatory network of differentially expressed genes.At the same time,genome-wide identification and characterization analysis of long non-coding RNA?lncRNA?in rice,basic characteristics,target gene prediction and functional analysis of lncRNA were carried out,and the heat response lncRNA?HR-lncRNA?of rice was screened.The main results was as following:1.A total of 18 samples of rice leaf tissue were used for ssRNA-seq?150 bp pair-end?,and each sample was sequenced to obtain clean data above 10 G.Cluster analysis of transcripts expression showed that the three replicates were consistent within each cultivar,which indicates the biological repeat is reliable.2.Differential expression analysis was performed on the gene expression profiles of ZS97 B and SYD2 after 2 and 6 days under heat stress,and a total of 3282 differentially expressed genes?DEGs?were obtained.Among them: 99 DEGs?GroupI?showed continuous up-regulation in two genotypes of rice,164 DEGs?Group II?showed continuous down-regulation in two genotypes of rice,and 291 DEGs?Group III?of specific up-regulation was shown in ZS97 B.3.GO and KEGG annotation and functional analysis were performed on the above three groups of genes?GroupI^III?.Genes of groupI were mainly enriched in GO term such as protein folding,responsive heat stimulation,nuclear transport and peptidase inhibitory activity;Genes of groupII were mainly enriched in GO term such as photosynthesis,transmembrane transport,lipid biosynthesis,phosphorus metabolism process,carbohydrate metabolism process and redox process;Genes of group III were mainly enriched in ribosomes and peroxisome/microbody process.4.The protein-protein interaction networks?PPI for group I^III?of the above three groups of genes were constructed separately,and the genes at the key nodes in the network topology were screened as the key regulatory genes for each group,including five key genes in group I?Os08g0326600,Os03g0831100,Os11g0703900,Os08g0487800,Os02g0115900?,six key genes in group II?Os04g0459500,Os12g0420200,Os04g0234600,Os01g0866400,Os08g0566600,Os11g0267000?and five key genes in group III?Os06g0274200,Os05g0552300,Os03g0350300,Os07g0673100,Os03g0271400?.5.A total of 2743 lncRNA were predicted in this study,which were classified into 4 types based on physical location,including 1408 intergenic lncRNA,323 antisense lncRNA,960 sense lncRNA,and 52 intron lncRNA.Of these,532 were highly similar to known rice lncRNA sequences.2211 are newly identified in this study.The basic characteristics of lncRNA analysis showed that lncRNA was evenly distributed in the genome without chromosomal bias.Compared with mRNA,lncRNA expression was low,transcript length was short,exon number was low,and homology analysis showed lncRNA sequeces among species was lowly conserved.6.The target genes were predicted for 1832 of 2743 lncRNA successfully.GO and KEGG enrichment analysis of target genes indicated that lncRNA were widely involved in carbohydrate,amino acid metabolism and lipids metabolism processes,as well as biological processes,response to stimuli and photosynthesis.Many target genes were also enriched in protein translation,folding processing,etc.,which indicates that lncRNA have potential roles in regulation at the translational and post-translational level.7.A total of 31 lncRNA closely related to rice heat stress response?HR-lncRNA?were screened,and for 16 of which target genes were successfully predicted.Many target genes were previously confirmed to play essential regulatory roles in heat stress.For example,the target gene?Os02g0711300,Os01g0337900?of TCONS₀0092993 are involved in the maintenance of redox homeostasis under heat stress,and the target gene?Os06g0195800?of TCONS₀0100154 is an important heat shock protein.The target gene Os01g0719100 of TCONS₀0005591 is involved in the regulation of stomatal opening under heat stress in rice.These findings significantly advance our understanding of the genetic basis of rice adaptation to heat stress and provide a substantial gene and lncRNA resources to the genetic improvement of rice.