Molecular Mechanism Of Abnormal Lignin Deposition Hindering Root Expansion Of Continuous Cropping Rehmannia Glutinosa

Rehmannia glutinosa L(R.glutinosa),a perennial herb of the Scrophulariaceae family.It is used as root medicine and is a well-known and widely used medicinal material in China.In the production of R.glutinosa,the problem of continuous cropping obstacles is very prominent,mainly manifested in the aggravation of diseases and insect pests,and the roots of the ground cannot expand normally.A large number of studies have shown that cell wall lignification,as an important means for plants to respond to external stress,significantly affects cell proliferation and growth.Based on this,this study uses the R.glutinosa variety "Wen 85-5" as the test material,and the key difference accumulation period of lignin synthesis is locked by measuring the dynamic difference accumulation of the first crop and continuous cropping R.glutinosa biomass and root lignin content;The differential expression analysis of the node genes in the lignin biosynthesis pathway at the transcription and protein levels,clarified the key node genes in response to continuous cropping stress;and the use of overexpression and interference expression reverse genetics to carry out the biological function of the key response gene RgCCR6 Verified.Below are key research findings:1.Spatio-temporal dynamic monitoring of lignin content in the roots of the first stubble and continuous cropping R.glutinosa and its deposition location.Based on the determination of biomass of the first crop and continuous cropping R.glutinosa,the dynamic determination of lignin content in the root crops of continuous cropping and continuous cropping by the acetyl bromide method showed that there was a significant accumulation process of lignin content in the root crops of continuous cropping and continuous cropping of R.glutinosa in 80 days.Compared with the first stubble R.glutinosa,it reached a very significant level of difference.In addition,except for 90 days,the lignin content in the roots of the continuous cropping R.glutinosa was higher than that of the first crop of R.glutinosa,and the tissue section observation using safranine solid green staining showed that the secondary xylem and central medulla of the continuous cropping R.glutinosa roots during 80 days A large number of lignified cells appeared between them,and these cells were arranged in a ring,which led to the obvious inhibition of the expansion process of the continuous cropping R.glutinosa roots.2.Identification of key node genes in response to continuous cropping stress in the lignin synthesis pathway.Using qRT-PCR technology to monitor the differential expression of 45 catalyzing enzyme node genes in the lignin metabolism pathway during the first crop and continuous cropping of R.glutinosa roots,six key genes in response to continuous cropping stress were preliminarily screened,namely RgC4H1,RgC3H2,RgCCo AMT7,RgCCR6,RgCAD2 and RgCAD5.These genes all showed significant expression characteristics in response to the poisoning of continuous cropping,especially in the 40-80 days after planting(seedling stage,pulling stage and pre-expansion stage).This study further cloned and antibody synthesized these six key genes,and used Western Blot technology to measure protein expression,showing that the expression of these proteins under continuous cropping is generally different from that of the first crop,while RgCCR6 is good for continuous cropping of R.glutinosa in the first crop.The differential expression in is particularly prominent.3.Biological function verification of key gene RgCCR6 in response to continuous cropping stress.By constructing overexpression vector and interference expression vector,Agrobacterium tumefaciens-mediated "leaf disc method" transformed R.glutinosa to obtain RgCCR6 genetic transformation positive strain,which was verified under continuous cropping simulation system.The results showed that the overexpressed plants had more fibrous roots,fresher roots,and lighter lignification than the interfering plants;by measuring the expression of the catalytic enzyme gene RgCCR6 at the transcription and protein levels,it was found that the overexpressing plants were compared to the control and interference expression The plants showed a significant upward trend.4.Reverse verification of catalytic enzyme inhibitors in the lignin biosynthetic pathway.Spraying key enzyme inhibitors PA and MDCA in the lignin biosynthesis pathway of the continuous cropping R.glutinosa seedlings artificially blocked the lignin synthesis pathway in the roots of R.glutinosa,and found that the lignin content of the continuous cropping soil was significantly reduced and the fresh roots The weight increased compared with the continuous cropping soil,but it was lower than the index of the first crop of R.glutinosa,and the effect of applying PA in the continuous cropping soil was better than that of MDCA.This study confirmed that the abnormal deposition of lignin significantly inhibited the process of root expansion of continuous cropping R.glutinosa from the physiological,biochemical and molecular levels,and initially locked the key catalytic enzymes of the lignin biosynthetic pathway in response to continuous cropping stress.The biological functions of key catalytic enzyme genes were verified,which provided an effective starting point for further research on continuous cropping obstacles.At the same time,it also provides a theoretical basis and technical support for further research on the stress stress and yield improvement of root crops.