| Rehmannia glutinosa L.(R.glutinosa)is a perennial herbaceous plant of Scrpophulariaceae with tuber roots.Recently,with the rising of traditional Chinese medicine health care,the demand for common Chinese medicinal materials has increased rapidly,such as R.glutinosa,which resulting in more and more economic benefits.Due to the limited land and the hazards of continuous cropping obstacles,the yield and quality of R.glutinosa were seriously reduced.Therefore,solving the problem of consecutive monoculture and improving the yield and quality of R.glutinosa are the common goals of many researchers.In this study,Wen "85-5"was used as the experimental material.The following studies were carried out with a view to Molecular Mechanism of consecutive monoculture of R.glutinosa.1.The screening and identification of specific response genes in R.glutinosa root.A.The screening of candidate genes.Based on the results of five different stress treatments(consecutive monoculture,ferulic acid,salt,drought,waterlogging)and five different stages(Seedling stage(stage I),Drawing period(stage II),Expansion Prophase(stage III),Expansion medium(stage ?),Later expansion period(stage V))of digital gene expression profiling(DGE),to screening of genes that specifically respond to consecutive monoculture.Compared with the first year plant(FP)of R.glutinosa,there were 2502,1956,2672,2485 and 7249 differentially expressed genes in five stresses respectively.A total of 678 differentially expressed genes were screened in second year plant(SP),to exclude the common differentially expressed genes of ferulic acid,salt,drought and waterlogging stress.Respective stage I as control for FP and SP,the FP has 3971,4403,3966,8190 differentially expressed genes in stage ?,stage ?,stage ? and stage V respectively,and the SP has 8595,7820,7792 and 12038 differentially expressed genes in stage II,stage ?,stage ? and stage V respectively.The contemporaneity of FP and SP has 7651,2090,600,3674 and 8433 differentially expressed genes respectively.4398 differentially expressed genes associated with SP were obtained by excluding genes with normal fluctuating expression levels during growth and development.By crossing 678 and 4398 specific genes selected from five stress treatments and five period treatments,80 common differentially expressed genes were obtained,functional analysis showed that some genes were involved in root cell secretion and endocytosis.It is preliminarily confirmed that candidate genes may be involved in the identification and transport of allelopathic autotoxic substances,thus producing a continuous disturbance response.B.The identification of candidate genes.Using the soil extracts from FP and SP soil to cultured seedlings,the responses of 49 candidate genes(|log2 ratio|?2 out of 80 candidate genes)to water-soluble extracts from soil around rhizosphere of R.glutinosa were detected.After repeated verification that there were 40 genes were accordance with the results of DGE in the seedlings cultured with the soil extracts from SP,and 27 genes had significant differences.To validate the consecutive monoculture response genes more accurately,we use ferulic acid,p-hydroxybenzoic acid and vanillic acid known as the key components of allelochemicals processing aseptic seedling respectively,and further analysis the 27 significant genes expression changing in the soil extracts from SP.The results showed that the expression level of some genes had a more severe fluctuation than that of soil extracts from SP under phenolic acid treatment.Finally,we locked in 10 genes to participate in the consecutive monoculture response of R.glutinosa,in which,CL2915.Contig2_All was up-regulation,Unigene32191_All,Unigene35559_All,Unigene29667_All,CL9586.Contigl_All,Unigene27064_All,Unigene35815_All,Unigene34664_All,Unigene18880_All and Unigene24_All were down-regulation.It's established the foundation for further study the function of these genes and their role in the consecutive monoculture of R.glutinosa.2.The identification of specific response miRNAs and obtain of silencing transformation plants.A.The identification of candidate miRNAs.Using qPCR to quantitatively identify the 21 miRNAs that were significantly different in the early screening of FP and consecutive monoculture,we accurately locked 9 miRNAs which specific response to consecutive monoculture,including 7 up-regulation(miR1074,miR530a,miR319d-5p.1,miR5054,miR1520d,miR2592,miR3951)and 2 down-regulation(miR156a,miR167a).According to the report,some of the miRNAs involved in stress and growth hormone signaling response,and miR5054 play an important role in MAPK signaling pathway and plant hormone signaling pathways.B.The creation of R.glutinosa genetic transformation system.After multiple experiments,we established a stable genetic transformation system.Firstly,using the leaf of R.glutinosa as explant to induce callus was better than that of root.Secondly,the MS medium which added 1.5mg/L 6-BA and O.lmg/L NAA had the highest callus induction rate,and the MS medium which added 2.0 mg/L 6-BA and 0.1mg/L NAA had the highest differentiation efficiency rate Thirdly,the optimized infectious concentration of Agrobacterium turnefaciens was OD600=0.6-0.8,and when the concentration of Timentin was 100mg/L,it will had a low rate of Agrobacterium tumefaciens contamination.Finally,when the screening concentration of Basta 0.2mg/L was the optimum screening strength.C.Obtain the miR5054 silencing transformation plants.The short tandem target mimic(STTM)vector was constructed and the genetic transformation system of R.glutinosa was established by leaf disk method,and the miR5054 silencing transgenic plants were successfully obtained.The establishment of stable genetic transformation system of R.glutinosa laid a foundation for further identification and analysis of miRNAs and genes related to the response of the consecutive monoculture of R.glutinosa. |
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