Transcriptome Analysis Of Sugarcane LncRNA And Functional Identification Of The Co-expression Gene ScTGA1 Under Sporisorium Scitamineum Stress

Sugarcane(Saccharum spp.)is an important economic crop in tropical and subtropical regions.Sugarcane smut caused by Sporisorium scitamineum greatly affects the yield and quality of sugarcane production.Breeding resistant varieties is one of the most important ways to solve sugarcane disease problems.Long non-coding RNA(lncRNA)do not encode any proteins,but can interact with DNA,RNA,and proteins,affecting transcription,post-transcriptional and epigenetic regulation events,and playing a key role in response to pathogen stress.However,the regulatory role of lncRNA in the interaction between sugarcane and smut pathogen has not yet been reported.The present study explored the expression profile of lncRNA in response to infection by the sugarcane S.scitamineum and analyzed the regulatory characteristics and functions of lncRNA in regulating the expression of target genes in sugarcane response to infection by the smut fungus.In addition,a positively regulated ScTGA1 gene,involved in pathogen infection,was cloned and identified from sugarcane cultivars ROC22.The aim of this study was to provide theoretical references for molecular breeding of sugarcane resistant to smut disease.The main results and conclusions of are as follows:1.The expression profile and regulatory network of lncRNA in sugarcane.During interaction between sugarcane ROC22 and S.scitamineum at 2 d and 5 d,a total of 21,270 lncRNA were predicted,of which 13,861 and 7,409 were identified in Saccharum spontaneum and Saccharum officinarum,respectively.The length of these lncRNA was mainly distributed between 200～1,000 nt,with 2～4 exons per lncRNA.WGCNA co-expression network analysis showed that 2,429 lncRNA were co-expressed with 49,887 genes,including 1,279 transcription factors.By screening resulted in 16 core lncRNA and 19 co-expression transcription factors.In addition,through the co-expression network analysis of lncRNA target regions,444 lncRNA and 372 target genes were identified to have cis-regulatory relationships,and 32 lncRNA were found to interact with the 21 core transcription factors.GO and KEGG analyses of cis-regulated target genes demonstrated that most of them are involved in flavonoid and flavonol biosynthesis,secondary metabolite metabolism,plant hormone signal transduction pathways,stress response,hormone signaling,enzyme families,and other functions.This suggests that lncRNA may play a regulatory role in antioxidant defense,growth and development,and disease resistance.Finally,seven groups of lncRNA with regulatory relationships and co-expressed genes were selected for RT-qPCR validation,and the results showed that the expression trends of RT-qPCR and RNA-seq were consistent,indicating that the sequencing results were reliable.2.Sugarcane ScTGA1 gene positively regulated plant defense against pathogen infection.Based on the results of the WGCNA co-expression network,we identified six lncRNA and the core co-expression gene Ss TGA1a/e that were involved in regulation.We successfully identified 44 Ss TGA genes in the genome of S.spontaneum.This gene belonged to the TGA gene family subfamily D,with Ss TGA1a/e belonging to clade II.Analysis of the cis-acting elements in the promoter regions revealed that the Ss TGA gene family had a large number of growth-related and hormone-responsive cis-acting elements.The Ss TGA gene family was constitutively expressed in different tissues of sugarcane and is induced by pathogen infection.Furthermore,we cloned a nuclear-localized homolog of Ss TGA1a/e from the sugarcane cultivar ROC22,named ScTGA1(Gen Bank accession number: ON416997).The c DNA length of this gene was 1,719 bp,with a coding region length of 918 bp encoding 305 amino acids.BLAST analysis revealed that ScTGA1 belonged to the typical b ZIP superfamily of genes.Hormone treatment of sugarcane demonstrated that ScTGA1 gene responded to salicylic acid(SA)and methyl jasmonate(Me JA).Compared to the wild-type control,tobacco(Nicotiana benthamiana)leaves overexpressing ScTGA1 showed a phenotype with smaller lesion areas and less accumulation of ROS.By upregulating the expression of tobacco immune pathway(HR,SA,JA,and ET)related genes,the ScTGA1 gene enhanced the resistance of tobacco to Ralstonia solanacearum and Fusarium solani var.coeruleum.This study provides some reference for the mechanism of lncRNA-target gene function in response topathogen infection.In summary,for the first time,this study systematically analyzed the lncRNA expression profile of sugarcane in response to S.scitamineum infection,analyzed the expression characteristics of sugarcane lncRNA,and constructed a regulatory network of lncRNA and target genes,and also mined the core lncRNA and target gene regulatory network in response to S.scitamineum infection.Besides,based on the S.spontaneum genome,we successfully identified 44 Ss TGA genes,and found that most family members participated in plant growth and development and actively responded to hormone and S.scitamineum infection stress.In addition,we successfully cloned a core nuclear gene ScTGA1 from sugarcane cultivar ROC22,which belonged to the typical b ZIP superfamily gene and can actively respond to SA and Me JA stress.The transient overexpression of sugarcane ScTGA1 gene in tobacco showed that the gene can positively regulated the resistance to pathogen inoculation by participating in HR,SA,JA,and ET signaling pathways.This study is expected to provide a reference for comprehensively understanding the regulatory network of sugarcane lncRNA,and for in-depth analysis of lncRNA molecular functions.At the same time,it also for provided potential gene resources with application value for breeding sugarcane varietyresistant to smut disease,which was of significant theoretical and practical importance.