Identification Of The Crucial Developmental Stage Of Rehmannia Slutinosa Under Consecutive Monoculture Based On Gene Expression And Tissue Culture Analysis

Rehmannia glutinosa L.(R.glutinosa),a perennial herb of Scrophulariaceae family,is one of the most common medicinal materials in China.The root tuber of this species have huge market demands.However,as an annual herb,the cultivation of R.glutinosa is limited by consecutive monoculture obstacle.Therefore,R.glutinosa often shows growth dwarfed and swelling inhibited under consecutive monoculture.In addition,replant diseases resulted in a poor harvest in both quality and yield obviously,even failure,which restricted the development of traditional Chinese medicine industry and local economy.Hence,consecutive monoculture obstacle of R.glutinosa has become an urgent problem to be solved.In order to reveal the molecular mechanism of consecutive monoculture obstacle,this study used “Wen 85-5”(R.glutinosa)as an experimental material and performed a series of research as follows.1.The analysis of differentially expressed genes in five developmental stages of first year planting(FP)and second year replanting(SP).This research group consider that the initiated fibrous roots and the initiated tuberous roots are the key stages according to the cell anatomy and phenotype analysis.The formation of the vascular cambium in the initiated fibrous roots stage,suggesting that this stage is the critical times for the initiation of the swelling of fibrous root and the responding to consecutive monoculture.To investigate the molecular mechanism of R.glutinosa growth and the critical times of the responding to consecutive monoculture,this study used Digital Gene Expression Profiling(DGE)to analyze adventitious roots(ARs),initiated fibrous roots(IFRs),initiated tuberous roots(ITRs),medium tuberous roots(MTRs),unexpanded fibrous roots(UTRs)of FP and SP,respectively.The results are as follows:A.The number of differentially expressed genes.The longitudinal and crosswise comparison has been carried out for collecting differentially expressed genes in FP and SP.The crosswise comparison used the ARs as control in FP and SP,respectively,and get the differentially expressed genes in four other stages.The longitudinal comparison screening to 3917,4403,3966 and 8190 differentially expressed genes in turn in FP R.glutinosa.Compared with ARs,MTRs(4403)and LTRs(8109)presented more differentially expressed genes than two other stages.Undoubtedly,the former related to expansion,the later related to the synthesis and accumulation of reserve materials(included medical component).The longitudinal comparison screening to 8595,7820,7792 and 12038 differentially expressed genes in turn in SP R.glutinosa.Obviously,a large number of genes showed differential expression in IFRs and LTRs two stages.Besides,the most significant distinguish between FP and SP were found in IFRs during different development stages.FP(3971)and SP(8595)was quite different by 216%.Hence,the gene expression has taken dramatic changes from ARs to IFRs in R.glutinosa under consecutive monoculture.The crosswise comparison is compared to each other in the same stage between FP and SP.Five stages screening to 7651,2090,600,3674 and 8433 differentially expressed genes,respectively,which indicated the ARs and LTRs had more gene expression perturbed.Taken together,the key stage of responding to consecutive monoculture obstacle is ARs and IFRs,which former than the key stage of swelling.B.Functional analysis of differentially expressed genes.GO and KEGG significant enrichment analysis of ARs and IFRs in FP and SP R.glutinosa showed that consecutive monoculture stress lead to biofilm sensitive,change the permeability of cell membrane and membrane receptor as well as reduce the activity of enzyme.More importantly,these changes above are likely to cause an array of interference,e.g.the inhibition of transduction and transports,the regulation of biological processes occur disorder and metabolic system disorders,etc.Compared with the gene expression of ARs and IFRs,it showed that differentially expressed genes significant enriched in cyclic nucleotide-gated channels and brassinosteroid synthetic pathway,speculating that the above-mentioned may be involved in the response of consecutive monoculture.Moreover,the DNA replication,RNA transport,protein synthesis and various enzyme system in R.glutinosa has also been damaged by consecutive monoculture.Clustering analysis of gene function further confirmed that the ARs and IFRs were critical times for responding to consecutive monoculture stress in R.glutinosa.2.The influences of soil extraction to aseptic seedling of R.glutinosaMost studies suggest that consecutive monoculture obstacle is caused by the roots secretion of autotoxins.However,as the soil nutritional condition is complex,coupled with the inference of microbial activity,it caused a huge challenge to analyzing.In order to get rid of the above-mentioned disadvantageous factors,this study simulated the harm of consecutive monoculture obstacle in a sterile condition and used the soil extraction from FP and SP soil(Most of the allelochemicals are water soluble organic acid)which has removed its bacteria.According to the medium(1 part soil extraction and 5 parts MS),observing the influence on plant growth.The analysis results are as follows:A.The influence of consecutive monoculture soil extraction to the growth of R.glutinosa seedling.According to growth and life cycle of R.glutinosa in culture medium,locking the critical value of root growth and expansion is 10 to 15 days.The R.glutinosa cultured in FP soil extraction as control of seedling and the other one as treatment,the aseptic seedlings have prominent phenotypic variation and the outstanding inhibition of root expansion after 10 days.This result indicated that the consecutive monoculture obstacle has appeared in the early stage of growth,which further confirmed the seedling R.glutinosa has already responded to the consecutive monoculture obstacle.B.The influence of consecutive monoculture soil extraction to calcium ion distribution of R.glutinosa seedling.Using the fluo-3 fluorescence indicator to track the soil extraction of different years to deal with the root tip of calcium distribution shows that the concentration of calcium ion increase remarkably in two years' consecutive monoculture of root tip.The result suggested that seedling root fluorescent determination of calcium ions can be applied to morphological index of autotoxins in R.glutinosa.