Identification And Functional Analysis Of Long Non-Coding RNA On Salt Tolerance Of Tamarix Hispida

Long non-coding RNA(lncRNA)is a class of transcripts which are larger than 200 nt in length and have extremely low protein-coding ability or without protein-coding ability.Previous studies have found that lncRNA in plants has regulatory functions in plant vernalization,floral transformation,reproductive growth,stress response,auxin transport,photomorphogenesis and lateral root development.LncRNA is one of the key regulatory in plant stress response.In this study,RNA-seq was used to sequence the whole genome lncRNA of Tamarix hispida in roots and leaves under salt stress for 6,24 h and non-stress conditions.A total of 13,622 lncRNA were identified,including 1,976 lncRNA in response to salt stress.In the root and leaf tissues of T.hispida under different salt stress time,the number of differentially expressed genes(DEGs)between the experimental group and the control group was calculated.The results showed that in leaf tissue,there were 1,039 DEGs in 0 h-VS-6 h and 1,039 DEGs in 0 h-VS-24 h;in root tissue,there were 2,009 DEGs in 0 h-VS-6 h and 2,448 DEGs in 0 h-VS-24 h.The differentially expressed lncRNA in root and leaf tissues of T.hispida under different salt stress time were classified by Gene Ontology(GO)terms and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway.The results showed that the highest proportion of genes were ’catalytic activity’,’binding’ and ’biosynthesis of amino acids’.To predict the function of the target gene of ThSAIR1-5(salt induced RNA),one target gene in ThSAIR4 and ThSAIR2 is NAC transcription factor,which can respond to salt stress.Five salt induced RNA(ThSAIRs)in response to salt stress were identified in this study.The expression specificity of T.hispida in leave and root tissues in response to salt stress were analyzed by quantitative real-time PCR(qRT-PCR).The results showed that the expression of five genes in leaves and roots changed significantly at different time points,indicating that all the five genes responded to salt stress and their responses to salt stress were preliminarily identified.The overexpression vector of ThSAIRs gene(pROKII-ThSAIR1-5)was constructed and transformed with the control empty pROKII vector by Agrobacterium tumefaciens-mediated transient transformation system,and the transient expression plants of T.hispida were obtained.Under salt stress,the physiological and biochemical indexes such as electrolyte leakage,water loss,malondialdehyde content(MDA).proline content(PRO),peroxidase(POD),superoxide dismutase(SOD)activity,Evans blue,diaminobenzidine(DAB)and nitrotetrazolium(NBT)staining of ThSAIR1-5 transient overexpression and control plants were analyzed to identify the salt tolerance function of ThSAIR1-5 gene.The results showed that overexpression of ThSAIR1-5 in T.hispida could significantly reduce the electrolyte leakage,water loss rate and MDA content,and increase the proline content;and the POD and SOD activities of ThSAIR1-5 overexpression plants were significantly higher than those of the control plants under salt stress.The above results show that under salt stress,the overexpression of ThSAIR1-5 can reduce the tissue cell damage and death of T.hispida,thus enhance the ROS scavenging ability of T.hispida,and then improve the salt tolerance of T.hispida plants with transient expression.Under salt stress for 24 h,200 DEGs were identified by RNA-seq of overexpressed ThSAIR5 and pROKII.The DEGs between the experimental group and the control group were analyzed by GO classification,KEGG pathway and enrichment analysis.The results showed that the highest proportion of DEGs were mainly involved in ’catalytic activity’,’binding’,’starch and sucrose metabolism’,and highly expressed in ’galactose metabolism’ and ’lysine degradation’.Through the prediction of overexpressed ThSAIR5 downstream target genes,it was found that peroxidase could encode proteins to respond to salt stress,and MYB and NAC transcription factors could respond to salt stress,indicating that ThSAIR5 gene could regulate the expression of some related genes in response to plant stress.