Functional Analysis Of LncRNA-RgAGT2 Promoter In Response To Consecutive Monoculture Obstacle Of Rehmannia Glutinosa L.

Rehmannia glutinosa L.(R.glutinosa)is a perennial herb in the family of scrophulariaceae.Its root has important medicinal value and it is one of the most famous Four Huai medicines in China.However,the cultivation of R.glutinosa has been limited by consecutive monoculture obstacle for a long time.Consecutive monoculture obstacle lead to the failure of normal expansion or even decay of R.glutinosa tubers,which seriously affects the yield and quality of R.glutinosa.Therefore,solving the problem of consecutive monoculture of R.glutinosa is of great significance to the sustainable development of R.glutinosa cultivation and production.In previous,our research group detected a long noncoding RNA(lncRNA)in response to consecutive monoculture obstacle through transcriptome sequencing.Because it is partially homologous with the coding gene of alanine glyoxylate aminotransferase 2(AGT2),it was named lncRNA-RgAGT2.It was found that lncRNA-RgAGT2 played an important role in the regulation of growth and development and stress response of consecutive monoculture obstacle R.glutinosa tubers.The research group cloned the 1088 bp upstream sequence of lncRNA-RgAGT2 by TAIL-PCR and RACE technology,which is speculated to be its promoter sequence(lnc-promoter).In order to further reveal the molecular mechanism of lncRNARgAGT2 in response to R.glutinosa consecutive monoculture obstacle,this study confirmed the transcription efficiency of 1088 bp sequence and dectected key ciselements and DNA methylation of lnc-promoter,which laid a foundation for further revealing the endogenous response network of R.glutinosa consecutive monoculture obstacle.The main results of this study are as follows:1.Based on the full-length sequence of lnc-promoter obtained by the previous work,the lnc-promoter::GUS expression vector was constructed by homologous recombination technology.Through Aagrobacterium mediated tobacco transient transformation and Arabidopsis thaliana transformation,we found that the reporter gene showed a high level of expression in different tissues of tobacco and A.thaliana.It indicated that lnc-promoter was the promoter sequence of lncRNA-RgAGT2.Considered that there was no significant difference in the transcription level of lncRNA-RgAGT2 in different tissues of R.glutinosa,lnc-promoter had no tissue specificity.2.Using PlantCARE to predict the cis-acting elements of lnc-promoter showed that the core cis-elements of TATA-box and CAAT-box were densely distributed in both the upstream and downstream of lnc-promoter,and the midstream contained ABA response element ABRE,low temperature response element LTR and other stress response cis-acting elements.The nc-promoter was truncated upstream of the IncRNARgAGT2 transcription start site.According to the truncation length,they were named P173,P447 and P826 respectively.P173::GUS,P447::GUS and P826::GUS expression vectors were constructed according to the above.The results showed that the activation efficiency of these truncated promoters were significantly lower than that of the fulllength promoter.Therefore,it was confirmed that P173 was the core promoter region of lnc-promoter and that the-826～1088 bp segment of the lnc-promoter was transcriptional enhancement region,which was prominent for its efficient activation.3.For preliminary locking lnc-promoter response consecutive monoculture obstacle cis-element applied exogenous stress(rhizosphere soil extract of consecutive monoculture(RSE),catalpol,IAA,H2O2 and ABA)to A.thaliana carrying different segmented promoters.After sampling and staining at different time points,it was found that P447 had a certain response to RSE,catalpol and H2O2,and P826 had a response to IAA and ABA.The preliminary results showed that the-174 to-826 bp segment located at upstream of the transcription initiation site was the main segment for lncpromoter to response stress.It was found that ABA had the most significant effect on the transcription efficiency of lnc-promoter compared with other treatments.Previous studies confirmed that consecutive monoculture obstacle induced change in ABA metabolism in R.glutinosa tuber roots.Considered that ABA treatment could induce significant up-regulation expression of R.glutinosa lncRNA-RgAGT2,it was comprehensively speculated that the ABRE located at-489 to-493 bp(antisense chain)upstream of the transcription initiation site may be the key element of stress response to consecutive monoculture obstacle.4.The genomic DNA of R.glutinosa was performed bisulfite sequencing.The results revealed that lnc-promoter had high-density cytosine methylation from-447 to-609 bp,and a large amount of demethylation occurred by consecutive monoculture stress,which may be caused by the high-level expression of active demethylase gene ROS1 induced by consecutive monoculture obstacle.After demethylation treatment using 5-azacytidine,the first planting R.glutinosa was detected a decrease expression in MET1,which is maintenance methyltransferase gene.Meanwhile,the lnc-promoter demethylation and lncRNA-RgAGT2 up-regulation were detected simultaneously in first planting R.glutinosa.In conclusion,through the analysis of 1088 bp sequence of lncRNA-RgAGT2 upstream in consecutive monoculture obstacle,this study confirmed that this sequence was a non-tissue-specific promoter,and preliminarily locked the ABRE,which could be the key cis-element for lnc-promoter to participate in the response to consecutive monoculture obstacle.Moreover,lnc-promoter was modulated by epigenetic modification,and lnc-promoter demethylation triggered by consecutive monoculture obstacle could improve lncRNA-RgAGT2 expression.