Function And Mechanism Studies Of Upland Cotton Long Non-coding RNAs-lncRNA354 And LncRNA973

Cotton is a moderately salt-tolerant economic crop,while salt stress affects the yield and quality of cotton,and the current research on the salt tolerance mechanism of cotton mainly focuses on physiological and biochemical levels and a few functional coding protein genes.Long non-coding RNA(lncRNA)is a class of functional RNA molecules with transcription length greater than 200 nucleotides without coding protein potential,which plays an im-portant role in plant response to biotic and abiotic stresses,the function and regulatory mech-anism of lncRNAs in cotton in response to salt stress is still unclear.In this study,two significantly differentially expressed lncRNAs were identified and screened from the previous laboratory transcriptome sequencing data.LncRNA973 was in-duced by salt stress,while lncRNA354 was inhibited by salt stress.The expression pattern in salt-stressed cotton and different tissues,molecular characteristics,as well as subcellular lo-calization and their regulatory mechanisms were analyzed.The main results were as follows:1.Molecular characterization and biological function analysis of lncRNA354.An inter-genic lncRNA-lncRNA354 whose expression was inhibited by salt stress was identified by RNA-seq,and it was located on chromosome A10 of upland cotton.Prediction and GUS test verified that it had no protein coding potential.Phylogenetic analysis found that lncRNA354was highly homologous on chromosome 10 of Asian cotton,and its neighboring genes in Asian cotton and upland cotton are homologous genes.In addition,lncRNA354 was localized in the nucleus and cytoplasm using nucleo-cytoplasmic isolation combined with q RT-PCR analysis.Using VIGS technology to silence lncRNA354 in upland cotton,the plant height,leaf area and root dry weight of lncRNA354 silenced plants were higher than those of control plants under salt stress conditions.The results suggested that lncRNA354-silenced cotton showed enhanced plants salt tolerance by inhibiting the accumulation of H₂O₂ and O^2-.2.Identification and validation analysis of lncRNA354 as miR160b mimic target.Using RNAhybrid software analysis,it was found that lncRNA354 could bind to multiple miRNAs,among which ghr-miR160b was significantly up-regulated in lncRNA354 silenced plants,and the expression level of its target gene ARFs was decreased.The miR160 family and its target genes,auxin-response factor ARFs,are highly conserved in plant evolution.Ghir?A05G042500(Gh ARF18)and Ghir?A06G004210(Gh ARF17)in upland cotton were identified as target genes of ghr-miR160b by GUS staining,q RT-PCR and 5'RLM-RACE.LncRNA354,mlncRNA354(mutated e TM site),ghr-miR160b and Gh ARF17/18-GUS were transformed into tobacco leaves individually or co-transformed transient expression,GUS staining and q RT-PCR analysis showed that the inhibitory effect of miR160b on Gh ARF17/18-GUS was co-expressed lncRNA354 blocked,while co-expression with mlncRNA354 failed to alter miR160b-mediated silencing of Gh ARF17/18-GUS.It was pre-liminarily determined that lncRNA354 could act as an endogenous mimetic target(e TM)of miR160b to regulate the expression of target gene ARFs.3.Function of lncRNA354-miR160b-ARFs regulatory network in cotton salt stress re-sponse.Based on TRV-mediated ghr-miR160b overexpression and silencing target gene Gh ARF17/18,the plant phenotype was similar to that of silencing lncRNA354,which pro-moted root development and improved plant salt tolerance.However,the upland cotton plants that silenced miR160b based on TRV-mediated STTM vector reduced salt tolerance.Ath-miR160 was significantly down-regulated in overexpressed lncRNA354 Arabidopsis plants.and the expression levels of target genes At ARF10,At ARF16 and At ARF17 were increased compared with the wild type,the salt tolerance of transgenic plants was reduced.However,the expression levels of ath-miR160 and target genes in overexpressed mlncRNA354 trans-genic plants remained unchanged,and their phenotypes were similar to those of the wild type.Overexpression of ghr-miR160b inhibited the expression of target genes At ARF10,At ARF16and At ARF17,promoted primary root development and inhibited the accumulation of H₂O₂and O^2-,and increased the salt tolerance of plants under salt stress,and its phenotype was op-posite to that of overexpressed lncRNA354 or the target gene Gh ARF17/18.4.Molecular characteristics and subcellular localization analysis of lncRNA973.LncRNA973 is a sense intergenic lncRNA,located on chromosome D04 of upland cotton and transcribed by RNA polymerase II.Its structure is similar to m RNA,both containing introns and exons.The results of coding potential analysis showed that lncRNA973 had no ability to encode proteins.LncRNA973 expression was increased in upland cotton under salt stress,and was highly expressed in root and cotyledon tissues.Phylogenetic analysis showed that lncRNA973 was a novel gene from G.raimondii with poor evolutionary conservatism among species.RNA FISH and nucleo-cytoplasmic separation methods were used to determined that lncRNA973 was mainly expressed in the nucleus and had weak signal in the cytoplasm.5.lncRNA973 may affect the expression of multiple genes to regulate upland cotton in response to salt stress.The full-length sequence of lncRNA973 transcript was cloned from upland cotton,and its overexpression vector was constructed to transform Arabidopsis to ob-tain OX-lncRNA973 transgenic Arabidopsis.OX-lncRNA973 transgenic Arabidopsis was treated with 100 and 150 m M Na Cl,and it was found that OX-lncRNA973 transgenic plants had increased salt tolerance compared with wild type.LncRNA973 was silenced in upland cotton by VIGS technology,compared with the control plants,the silenced lncRNA973 plants had reduced salt tolerance and affected the accumulation of some physiological indicators such as MDA,proline,and sodium and potassium ions.There was no significant change in expression level of lncRNA973 neighboring genes in VIGS plants,and it may respond to salt stress by regulating gene expression in trans.RNAplex and q RT-PCR were used to predict and verify that lncRNA973 may regulate the salt stress response of upland cotton by affecting the expression of salt stress-related genes such as RBOHB,P5CS,NHX7,RD22 and SOD,as well as transcription factors MYB5,WRKY46,NAC29 and ERF62.In summary,the lncRNA354-miR160b regulatory network affects cotton growth and de-velopment and response to salt stress,while lncRNA973 may positively regulate the cotton salt tolerance by affecting the expression of multiple salt stress-related genes.It is speculated that the two lncRNAs could coordinately regulate cotton growth and development and salt stress tolerance.The research on the function and mechanism of the two lncRNAs will deepen the understanding of the salt tolerance mechanism of cotton,and provide a theoretical basis for the identification of cotton salt tolerance and the selection of new varieties with salt toler-ance.