| Cotton（Gossypium spp.）is one of the important economic crops in the world.Although cotton has strong salt tolerance,salt stress has long been an important factor affecting cotton growth and development,threatening cotton yield and quality.Long noncoding RNAs（lncRNAs）are a class of functional RNA molecules,which participate in the regulation of different biological processes at multiple levels in the form of RNA in biological processes,and play an important physiological role in regulating plant growth,development and enhancing plant stress resistance.At present,there are only a few reports on the stress regulation function of lncRNA in cotton.In previous study,we identified lncRNAs related to salt stress in upland cotton,and verified that lncRNA973 was involved in the regulation of salt tolerance in upland cotton by virus-induced gene silencing（VIGS）and heterologous overexpression in Arabidopsis thaliana.However,the function of lncRNA973 was not studied by overexpression in cotton.In addition,the regulation of lncRNA973 expression in cotton was remains unclear.In this study,HM-1,a salt-sensitive upland cotton variety,was used as the research material,and the overexpressed lncRNA973 plants were obtained through genetic transformation.Then,the growth physiological indicators under salt treatment were detected,which verified that lncRNA973 positively regulated the salt tolerance of upland cotton.In addition,the cis-acting elements of lncRNA973 promoter were identified by analysis of lncRNA973 promoter.The main results are as follows:1.The hypocotyls of sterile seedling were used as explants to induce callus,and the lncRNA973 overexpression vector was introduced into salt-sensitive upland cotton variety HM-1 by agrobacterium-mediated genetic transformation.Callus infected with agrobacterium tumefaciens were screened on screening medium of kanamycin and regenerated transgenic seedlings were obtained by continuous subculture and rooting induction.Then,PCR detection of the selected marker gene Npt Ⅱ in transformed regenerated plants showed that 25 positive regenerated overexpressed lncRNA973 upland cotton plants were obtained.Meanwhile,fluorescence quantitative PCR analysis showed that the relative expression levels of lncRNA973 in these positive regenerated plants were significantly increased.2.Overexpression of lncRNA973 mitigates salt stress in upland cotton.By comparing the growth of upland cotton plants under salt treatment（250 mM NaCl）,it was found that the overexpression of lncRNA973 mitigated the growth inhibition caused by salt stress and enhanced the salt tolerance of HM-1.Physiological indexes of HM-1 and overexpressed lncRNA973 plants under salt stress were detected,and the results showed that overexpression of lncRNA973 increased the relative water content,chlorophyll,proline（Pro）,soluble sugar（SS）contents,the activities of superoxide dismutase（SOD）and catalase（CAT）and the content of reduced glutathione（GSH）of upland cotton seedlings leaves under salt stress,which maintained the metabolic level,osmotic regulation ability and scavenging ability of reactive oxygen species of upland cotton under salt stress,mitigated the growth inhibition caused by salt stress to upland cotton,and enhanced the salt tolerance of cotton.3.Functional analysis of lncRNA973 promoter in upland cotton.The full-length sequence of the lncRNA973 promoter（P＿lncRNA973）was cloned from upland cotton.Then,the expression vectors of different lengths of P＿lncRNA973 fused to GUS gene were constructed according to the position of each cis-acting element on P＿lncRNA973 and transformed into Arabidopsis thaliana.Next,each P＿lncRNA973 fragment in the transformed system was analyzed by GUS histochemical staining.Through promoter activity analysis,we found that the most active region in P＿lncRNA973 is the short fragment located between P2 and P1（-247 bp～-153 bp）at the 3’ end,which may be responsible for regulating the basic constitutive expression of lncRNA973 in a specific environment.There are inhibitory regulatory elements between P3 and P2（-342 bp ～-247 bp）with strong negative regulatory characteristics,and active regulatory elements between P8 and P6（-828 bp ～-581 bp）.Moreover,the recognition site（-1525 bp ～-1519 bp）of salt-induced cis-element MYCATERD1 in the promoter fragment between P15 and P14（-1536 bp ～-1429 bp）may be the key cis-element that enforces the salt-induced activity of P＿lncRNA973. |
