Functional Characterization Of LncRNA In Response To Salt Stress

As an important source of human protein and edible oils,soybean（Glycine max）plays an important role in agricultural production.However,the soybean industry China is now facing a continuous decline in self-sufficiency rate,drought and soil salinization and other stresses restricts the growth of soybean production greatly.Making full use of plant stress resistance genes to cultivate new high-resistance varieties is the most fundamental,economical and effective way to improve plant stress resistance.The excavation and functional study of stress-resistant soybean genes is an important basis for the cultivation of new soybean varieties.Long non-coding RNA（lnc RNA）usually refers to non-coding RNAs with a length greater than 200 bp,lacking an open reading frame,and no protein encoding ability.As an important part of the transcriptional regulatory network,lnc RNA plays an important role in plant stress response.However,the functional studies of lnc RNA in plants are still relatively rare.In the early stage,transcriptome sequencing of soybean roots treated under normal conditions and salt stress was carried out.Based on this study,two lnc RNAs with large fold change under salt stress and their antisense target genes were selected.A.rhizogenes K599 mediated soybean genetic transformation system has higher conversion efficiency,simple process,less pollution and short cycle than traditional genetic transformation methods.In this study,the selected lnc RNA and its target gene were cloned into p GFPGUS vector by homologous recombination method,and overexpressed by 35S.Then transformed into Agrobacterium rhizogenes K599 to form soybean transgenic composite.The function of lnc RNA was studied by analyzing the response of soybean transgenic composite to high salt stress.The follows are the main findings:The results of qRT-PCR analysis showed that:under salt stress,lncTCONS＿00072963 and its target gene Gm EGY1 were up-regulated and down-regulated,respectively;lncTCONS＿00042461 and its target gene Gm TFIIIC were down-regulated and up-regulated,respectively,consistent with the results of transcriptome sequencing analysis.2.Overexpression of lncTCONS＿00072963 in soybean hairy root:the salt tolerance of soybean overexpression composite under salt stress was higher than that of the vector control composite.In the following main aspects:DAB and NBT staining of the composite leaves showed that the H₂O₂ and O^2-content in the leaves of the overexpression composite was lower than vector control composite;the chlorophyll content was higher than vector control composite;The electrolyte leakage rate was lower than vector control composite.By measuring the malondialdehyde content,it was found that the peroxide content in the root of the overexpression composite was lower than vector control composite;the Na⁺/K⁺in the stem and the leaf was significantly lower than vector control composite.The above results indicate that overexpression of lncTCONS＿00072963 in soybean hairy roots significantly improved the salt tolerance of the transgenic composite.3.In the same way,the antisense target gene Gm EGY1 of lncTCONS＿00072963in soybean hairy roots was overexpressed,the salt tolerance of the overexpression composite was reduced,mainly as follows:H₂O₂ content and electrolyte leakage in leaves of transgenic composite under salt stress conditions was higher than vector control composite.While the chlorophyll content was lower than vector control composite;the Na⁺/K⁺in the roots,stems and leaves of the transgenic composite increased significantly;the MDA content in the root increased.The above results indicate that overexpression of the Gm EGY1 in soybean hairy roots significantly reduces the salt tolerance of the transgenic composite.4.Overexpression of lncTCONS＿00042461 in soybean hairy roots,under salt stress,the accumulation of H₂O₂ in leaves of transgenic composite increased,the chlorophyll content decreased,and the electrolyte leakage increased;Na⁺/K⁺in roots and leaves of overexpression composite was significant higher than vector control composite;the MDA content in the roots was higher than vector control composite.The above results indicate that overexpression of lncTCONS＿00042461 significantly reduced the salt tolerance of the transgenic composite.5.The antisense gene Gm TFIIIC of lncTCONS＿00042461 in soybean hairy roots was overexpressed,the salt tolerance of overexpression composite was enhanced,which is manifested in the following aspects:H₂O₂ content and electrolytes leakage in leaves of overexpression composite under salt stress conditions were lower than vector control composite,and the chlorophyll content and water content were higher than vector control composite;the Na⁺/K⁺and MDA content in the roots of overexpression composite were significantly lower than vector control composite.These results indicate that overexpression of Gm TFIIIC can significantly increase the salt tolerance of the transgenic composite.