| Rationales: Astragalus membranaceus(Fisch.)Bge.var.Monghlicus(Bge.)Hsiao,a large number of traditional medicinal materials in China,"This product is the dried root of the leguminosae A.membranaceus(Fisch.)Bge.var.monghlicus(Bge.)Hsiao)or A.membranaceus(Fisch.)Bge." It has the functions of tonifying qi and rising Yang,benefiting Wei solid surface,nourishing toxin and regenerating muscle,promoting water and detumescence.At present,the planting mode of A.monghlicus is mainly to imitative wild A.monghlicus and cultivated A.mongholicus..The traditional growth pattern of A.monghlicus is wild or mimicking wild,but it is difficult to collect and dig(root depth can reach 1 m)because it takes a long time to mature and harvest(generally 5-6 years)under this growth pattern.With the increasing demand for astragalus,it is urgent to develop a new cultivation method of astragalus.In the 1980 s,the flat planting method of A.monghlicus was developed,which not only reduced the planting cost,but also rapidly expanded the planting area of A.monghlicus,but also had a certain impact on the quality of traditional wild A.monghlicus.Our research group is located in Shanxi,one of the authentic producing areas of A.monghlicus,and has been engaged in the quality evaluation of A.monghlicus for a long time.Our previous research found that the changes of cultivation methods led to significant changes in its character,quality and efficacy.In terms of anti-fatigue and immune effects,the effect of cultivated A.monghlicus was weaker than that of wild type under different cultivation methods.In the secondary metabolites of commercial A.monghlicus,the isoflavones and methyl glycosides in the wild A.monghlicus were higher than those in the cultivated A.monghlicus,and the key enzyme genes in the molecular synthesis pathway were IFS,CHS,4CL and PAL.HMGR,MVD,HDR,and SS.Therefore,it remains to be further explored what important genes are involved in influencing the morphological characteristics of the main and lateral roots in A.monghlicus.At present,the molecular mechanism of the main and lateral root traits of A.monghlicus is still unclear.By reviewing a large number of Chinese and English literatures,we summarized the root growth and development genes of the dicotyledonous plant Arabidopsis thaliana,and tried to use molecular biology methods to obtain the genetic background and dynamic changes of transcriptome level of the two growth patterns of A.monghlicus.We analyzed the correlation between epigenetic traits and genetic basis,and explained the formation mechanism and biological nature of trait differences under different growth patterns.Based on this,in this study,based on the second and third generation transcriptome data of A.monghlicus obtained by the previous sequencing of our research group,we analyzed the transcriptome of 1-6 years old wild A.monghlicus and 1-2years old cultivated A.monghlicus samples,and further carried out the weight gene coexpression analysis.To screen the traditional Chinese medicine(TCM)genes affecting the main and lateral root system structure of A.monghlicus,and to observe the changes in the gene levels of A.monghlicus under different years and different growth patterns,in order to reveal the molecular regulation mechanism of the differences in main and lateral root traits between flat cultivation and imitation wild A.monghlicus,so as to promote the development of high-quality authentic A.monghlicus industry,classify and apply A.monghlicus,and improve the clinical effectiveness.Objective:1.Through trait comparison and morphological characterization,the differences of main and lateral root traits of A.monghlicus under different growth patterns were revealed from the macroscopic level.To screen the genes related to main and lateral root traits in flat cultivation and wild imitation of Astragalus mongolicus.To understand the molecular mechanisms controlling the growth and development of main and lateral root traits in flat cultivation and wild imitation of Astragalus mongholicus.Methods:1.At the macroscopic level,the root length,diameter and the number of lateral roots were measured and other appearance traits of the flat and wild type A.monghlicus were measured.One-way ANOVA was used to analyze the differences between the flat and wild type A.monghlicus samples.2.By making paraffin sections and safranin-fast green staining methods,morphological characteristics of 6-year-old wild A.monghlicus,2-year-old and 3-year-old flat-planted A.monghlicus,which are the main commodities circulating in the market,were carried out.By distinguishing the microscopic characteristics of the cell structure of the three kinds of plants,the main differences in the growth and development of the roots of A.monghlicus under the two growth patterns were revealed.3.By constructing the weighted gene co-expression network of A.monghlicus under different years and different growth patterns,gene modules were divided,and GO enrichment analysis and KEGG pathway analysis were performed on the module genes of interest.4.Based on the dicotyledonous model plant A.thaliana,the genes affecting the growth and development of main and lateral roots were summarized and sorted out by reviewing the literature.Combined with the second and third generation of A.monghlicus transcriptome data established by our group,the key genes that promoted the elongation and thickening of the primary root and the increase of lateral root density were screened by bioinformatics methods such as screening and filtering FPKM values,constructing phylogenetic trees,and comparing gene sequences.The predicted expression value(FPKM)of each gene was analyzed by Spearman correlation.5.The m RNA expression levels of key genes for primary root elongation,thickening and lateral root density of A.monghlicusin different growth patterns were explored by fluorescence quantitative experiment,and the results were analyzed by Spearman correlation analysis.6.Spearman correlation analysis was performed on the expression levels of the screened genes affecting the growth and development of the main and lateral roots of A.monghlicus and the key genes in the biosynthesis pathway of secondary metabolites such as isoflavones and methyl glycosides found in the previous research of our group.Results:1.The appearance traits of 6-year-old imitami-wild Radix astragali,2-year-old and 3-year-old radix planensis were measured.The results showed that the primary root length of 6-year-old imitami-wild A.monghlicus was significantly longer than that of Radix planensis,the number of lateral roots was significantly less than that of Radix planensis,and the diameter was similar.At the same time,the results of one-way ANOVA showed that there were significant differences in the main root length and the number of lateral roots among different sizes of A.monghlicus,but there was no significant difference in diameter.2.The results of morphological analysis showed that there were obvious differences between the 6-year-old wild type and the 2-3 year-old plain cultivated A.monghlicus.There were more cells in the wood fibers and bast fibers.Wood rays and phloem rays were more closely arranged.The xylem and phloem duct cells were clustered in 30 to 50 bundles,closely arranged,and the xylem had many radiation angles.There were fewer cells in the cork layer.3.The weighted gene co-expression network was divided into 9 modules,among which the modules related to wild A.monghlicus and Radix planatus membranaceus were black modules.GO enrichment analysis of genes in the modules showed that they were concentrated in the cellular amino acid synthesis pathway,and KEGG pathway analysis showed that they were concentrated in the ubiquitin-mediated proteolysis pathway.4.Through the screening of the main and lateral root related genes of A.thaliana and the multiple amino acid and gene comparison with the A.monghlicus transcriptome database by bioinformatics methods,the gene HRS1,WOX4 and KNAT3 affecting the main root elongation,the main root thickening and the lateral root growth and development of A.monghlicuswere screened.5.Through the fluorescence quantitative experiment of the key genes related to the main and lateral roots of A.monghlicus,the expression of the above genes was preliminarily verified to be one of the reasons leading to the differences in main and lateral root traits between the flat cultivation and the wild type of A.monghlicus.The Spearman analysis of FPKM values showed that the main root elongation gene and the main root thickness gene r=0.6819(p<0.05).There was a significant correlation between them.6.Spearman correlation analysis was performed on the expression levels of the related genes affecting the root traits of A.monghlicus and the key genes in the biosynthesis pathway of secondary metabolites such as isoflavones and methyl glycosides,and the results showed that: The primary root length HRS1 and enlargement WOX4 genes were positively correlated with isoflavones 4CL,C4 H,MVD and HDR genes(r > 0.6),while the KNAT3 gene was negatively correlated with isoflavones HMGR,SS,MVD and HDR genes(r >-0.6).Conclusions: At the macro level,this study characterized the differences in appearance traits and morphology between imitative wild A.mongholicus and cultivated A.mongholicus.At the gene level,HRS1,WOX4,and KNAT3 were identified to affect the main root elongation,the main root enlargement,and the lateral root growth and development under different growth patterns,respectively.The results of preliminary validation indicated that these genes might be one of the reasons for the differences in main root and lateral root traits.With the increase of primary root elongation and thickened gene expression,the gene expression of secondary metabolites such as astragalus mongholicus isoflavone and methyl glycoside increased. |
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