Cloning And Preliminary Functional Analysis Of LncRNA-RgAGT2 Responding To Consecutive Monoculture Obstacle In Rehmannia Glutinosa L.

In recent years,the demand for Chinese medicine has increased because of the rise of traditional Chinese medicine.Rehmannia glutinosa L.(R.glutinosa)is one of the most important medicinal materials in China which is exported to home and abroad.However,in its cultivation and planting,there are serious continuous cropping obstacles,which greatly affects the yield and quality of Rehmannia glutinosa.The search for its molecular mechanism is one of the important issues in the research field of R.glutinosa.This study was based on the previous response RNA-Seq screened for the unique response to the continuous disorder transcript Unigene29334_All,and cloned the complete transcript with a length of 1573 bp using hiTAIL-PCR and RACE techniques.The ORF Finder analysis showed that all the ORFs of the cloned products were less than 300nt,and the transcript was confirmed to be long non-coding RNA(LncRNA).In comparison with the R.glutinosa transcriptome database,this LncRNA is homologous to a portion of the gene coding for alanine glyoxylate transaminase 2(RgAGT2).Therefore,the LncRNA was cloned and named LncRNA-RgATG2.To further study the function of LncRNA-RgAGT2,we examined the expression patterns of LncRNA-RgAGT2 and RgAGT2 genes in the five critical periods(seedling stage,Drawing period,Expansion Prophase,Expansion medium,Later expansion period)of growth and development of the first year plant(FP)and the continuous cropping of R.glutinosa.The results suggest that LncRNA-RgAGT2 may have a regulatory relationship with RgAGT2 gene in R.glutinosa.At the same time,LncRNA-RgAGT2 exhibited significant fluctuations in all stages of R.glutinosa,and it can be used as a"marker gene" for the early diagnosis of continuous cropping yellow.For the first time,this study found that LncRNA participates in the response and regulation process of R.glutinosa,and it is a powerful complement to the molecular mechanism of rehabilitative stem disease in R.glutinosa.In order to further study the role of LncRNA-RgATG2 in the continuous cropping disorder of R.glutinosa,we constructed the RNAi silencing vector of LncRNA-RgATG2 and obtained the positive plants of LncRNA-RgATG2 silence by Agrobacterium-mediated leaf disc transformation.Based on the previous work of regulation of hormones concentration induced expansion of R.glutinosa roots cultured in MS medium,this work treated the positive plants of LncRNA-RgATG2 silence with(6-BA and NAA)to induce expansion of roots,and observe the effect of hormones on root enlargement of LncRNA-RgATG2 silent plants.The results showed that both the uninfected regeneration seedlings and the LncRNA-RgATG2 silenced plants were expanded under hormone treatment,and the LncRNA-RgAGT2 and RgAGT2 genes were down-regulated,but the roots of LncRNA-RgATG2 silenced plants were significantly larger than the regeneration seedlings.At the same time,hormone treatment accelerated the expansion of LncRNA-RgATG2 silenced plants compared to the expanded LncRNA-RgATG2 silenced plants on hormone-free normal rooting medium.It is speculated that LncRNA-RgATG2 is involved in the expansion of the roots of R.glutinosa and is related to hormones.It may be involved in the regulation of R.glutinosa roots enlargement through the hormonal signal response pathway together with RgAGT2.In this study,we explored the role of LncRNA-RgATG2 in the continuous resistance of R.glutinosa by analyzing the function of LncRNA-RgATG2 in response to the obstacles of R.glutinosa,thereby further revealing the molecular mechanism of the continuous resistance of R.glutinosa.