The Screening And Functional Analysis Of The Key Genes Responding To Continuous Monoculture Problems During The Development Of Rehmannia Glutinosa Libosch

Rehmannia glutinosa Libosch(Rglutinosa),a perennial herbaceous medicinal plant,belongs to the Scrophulariaceae family.R.glutinossa is one of the famous "Four Huaiyao" and has a long history of cultivation.However,its productivity and quality declines seriously when it is grew on the field where the preceding crops were themselves,a phenomenon described as "continuous monoculture problem",the field can replant R.glutinosa at an interval of eight to ten years,which severely restricts the production of R.glutinosa and sustainable use of land resources.Currently,the studys on the causes of continuous monoculture disease are focused on soil microorganisms,autotoxicity of root exudates and its physiological activity,etc.,but the molecular mechanism of its formation is unclear.In this study,we use"Wen 85-5",one of R.glutinosa varieties as experiment materials,continuous cropping(R.glutinosawas planted second yearin the same-land,SP)as treatment,and the first planted(R.glutinosawas planted first year in a land,FP)as control.Then,samples of roots were captured at the representive periods of R.glutinosa development,including seedling stage,root strecth stage,the initial stage of root enlargement,the mid stage of tuber enlargement,and maturity stage.On that basis,we construct transcriptome library for these ten samples that includedroots and leaf of continuous cropping and normal cropping Rglutinosa,through using a new generation of Illumina sequencing technology,and integrate them with the previous transcriptome;meaningwhile,RNA-Seqtechnology was used to build different digitized profiles in gene expression in the different developmental stages of R.glutinosa roots,to screen key genes responding to the formation of continuous monoculture disease,and to clarify the molecular mechanism of continuous monoculture disease.The main findings are as follows:1.The samples of R.glutinosa root(R)and leaf(L)were sequenced by Illumina technology,and from every sample we get more than 12,000 millions raw reads,after assembled,roots and leaves respectively obtained 71,922unigene and 130655 contigs,54,722 unigene and 102,253 contigs,then we assembled them with the transcriptome our research group obtained before,we totally got 66,960 unigene.Homology comparison ofthe unigene,swissprot,Nr protein libraries and other relevant databases above show that,in Nr database,there are 46,147 sequences having a certain degree of homology with the functional genes of other species;and that there are 30,947 sequences used for comment in the Swissprot database;COG classification shows there are 18,029 sequences that have functional annotation;GO classification shows there are 34,912 sequences that have molecular function;KEGG analysis shows there are 27,532 sequences which involve in 119 metabolic pathways.Under the condition of the R.glutinosa genome is unknown,we established and integrated all the genetic information of Kglutinosa in this study,which laid the foundation of technology and information for revealing the molecular mechanism of continuous cropping obstacles.2.The screening of the key genes responding to continuous cropping problem in different growth stages:We contructed the digital gene expression profiling of continuous cropping and first cropping Rglutinosa roots in the five growth stages(seedling stage,root strecth stage,the initial stage of root enlargement,the mid stage of tuber enlargement,and maturity stage),every library gets more than 6M raw reads.We make continuous cropping R.glutinosa as treatment,the first planted R.glutinosa as control,and analyze critical genes responding to the harmless of continuous cropping in different growth stages.The results show that:There are 7,653 differentially expressed genes screened between continuous cropping and first cropping R.glutinosa(SP1/FP1)in the seedling stage;602 differentially expressed genes were screened in the root strecth stage;2,092 differentially expressed genes were screened in the initial stage of tuber root enlargement;3,676 differentially expressed genes were screened in the mid stage of tuber enlargement;8,435 differentially expressed genes were screened in the maturity stage.The function analysis on genes responding to continuous cropping in the five key periods shows that:In the continuous cropping R.glutinosa,the modification mechanism of chromatin was inhibited,the key process blocked cell division,and the plant growth and development related processes being completely broken in seedling stage;hormone metabolism was disordered,the salicylic acid-related genes being significantly up-regulated,growth of root was inhibited in the root stretch stage;the perception of R.glutinosa to light being affected in the initial stage of tuber root enlargement;salicylic acid signaling pathway is extremely significant increasing in the mid stage of tuber enlargement;The aboveground part of continuous cropping R.glutinosa being basically dead,part of the root re-developping into seedlings in the maturity stage.To sum up,the poison of continuous monoculture was perceived in the seedling stage,and the hazard is gradually increasing with the promotion of the development process.3.The study on differences of development pattern between continuous cropping and first cropping Rglutinosa:We made the first cropping and continuous cropping R.glutinosa in the seedling stage as the control respectively,made the R.glutinosa in root strecth stage,the initial stage of root enlargement,the mid stage of tuber enlargement,and maturity stage as treatment then we analyzed the differences of development pattern between continuous cropping and first cropping R.glutinosa.Results are as follows:In first cropping R.glutinosa,compared with seedling stage,in root pull phase stage,the initial stage of root enlargement,the mid stage of tuber enlargement,and maturity stage we respectively obtained 4,405,3,973,3,968,8,192 differentially expressed genes;in the continuous cropping Rglutinosawc respectively obtained 7,822,8,597,8,794,12,040 differentially expressed genes in the five stages.The analysis on function of key differently expressed genes respectively in the development process of continuous cropping and first cropping Rglutinosa shows that:in the development process of first cropping R.glutinosa,a large number of genes which are closely related to cell cycle regulation,protein synthesis and development are concertedly and orderly expressed;while in continuous cropping R.glutinosa,the order of the considerable genes that are related to development and metabolism is completely disrupted,and genes associated with chromatin modification in continuous cropping R.glutinosa were significantly upregulated.This suggests that in the poison process of continuous cropping R.glutinosa,a large number of genes are turned on or turned off.Under the condition that R.glutinosa genetic information is scarce at present,in this study,we constructed the transcriptome library of its root and leaf,established digitial differentially expressed profiling for continuous cropping Rglutinosausing new-generation sequencing technology,and screened the key genes responding to continuous monoculture disease.The results from different experiments,formed a complete chain of evidence:Toxic effects of continuous cropping include disordering the normal gene expression in continuous cropping Rglutinosa through inhibiting the chromatin modification mechanism,which further closed leading to cell cycle process being closed,and metabolic disruption,and at the same time,light response were also significantly suppressed in contious cropping R.glutinosa.In addition,salicylic acid and other hormone promoted aging were substantially expressed in continuous cropping R.glutinosa.This study outlines the "poison" mechanism of continuous cropping R.glutinosa,which has laid a theoretical foundation for studying the causes and molecular mechanism of continuous cropping obstacle in R.glutinosa,and developing an effective technology to reduce the harm of continuous cropping.It also provides a beneficial reference for the study of molecular mechanism of continuous cropping obstacle on other plants.