Study On The Mechanism Of Morphological Variation Based On RNA Sequencing In Atractylodes Lancea

Atractylodes lancea(Thunb.)DC.,a perennial plant of Compositae,belongs to traditional Chinese medicine in China.At present,the main producing areas of Atractylodes lancea on the market have moved from Maoshan,Jiangsu Province,a historical genuine producing area,to the artificial large-scale planting bases in Yingshan,Luotian and other counties of Huanggang City,Hubei Province.Atractylodes lancea(Thunb.)DC.The ramets degenerated and changed,forming a variety of variation types,including the flat stem variation of Atractylodes lanceolata.The quality differences of Atractylodes lanceolata and Atractylodes lanceolata var.lanceolata were studied from the aspects of pathogen,chemical composition and transcriptome.The aim is to elucidate the mechanism of variation of rhizoma atractylodis rhizome and improve the quality of cultivated medicinal materials.The main contents of this paper are as follows:1.Identification of pathogens causing flat stem variation of Atractylodes lancea by molecular biology and histochemistry.Combining the morphological characteristics of Atractylodes lanceolata,it was found that the symptoms of disease caused by phytoplasma were consistent.Therefore,the existence of phytoplasma in Atractylodes lanceolata var.lanceolata var.lanceolata was detected by nested polymerase chain reaction using the universal primers of 16 S rRNA gene of phytoplasma.Through gene sequence analysis and phylogenetic analysis,it was clear that the phytoplasma detected in Atractylodes lanceolata var.hupekinensis belonged to 16SrI-B PHYT The subgroup provided scientific basis for the prevention and control of phytoplasma diseases of Atractylodes lanceolata.Through the later verification of DAPI staining,compared with the normal Atractylodes lanceolata,a large amount of dot fluorescence was found in the transverse section of the stem of the variant plant,which further verified the existence of phytoplasma in Atractylodes lanceolata.At the same time,the severity and distribution of the disease can be known by statistics of the incidence of the disease in four bases of Huanggang City.2.The effect of flat stem variation on yield and quality of Atractylodes lanceolata was explained by the related indexes of yield and quality.According to the description of Atractylodes lanceolata entries in the Chinese Pharmacopoeia 2015 edition,the comparison between Atractylodes lanceolata and control Atractylodes lanceolata was made in terms of character,microscopy,TLC and content determination.The results showed that Atractylodes lanceolata var.lanceolata var.lanceolata var.lanceolata var.lanceolata var.lanceolata var.lanceolata var.lanceolata var.lanceolata var.lanceolata var.The volatile oil of Atractylodes lanceolata is dark yellow,while that of Atractylodes lanceolata is light yellow,the average content of volatile oil in atractylodes plantarum was 10% higher than that in normal atractylodes plantarum.There were only slight differences in microscopic identification,water content,TLC and beta-eucalyptol content.The chemical constituents of Atractylodes lanceolata and Atractylodes lanceolata were analyzed in order to provide references for the correct recognition and utilization of Atractylodes lanceolata resources.3.Study on the variation mechanism of Atractylodes lanceolata flat stem based on RNA-seq.Twenty-four samples of flowers,leav es,stems and rhizomes of Atractylodes lanceolata and Atractylodes lanceolata were sequenced using Illumina Hi-Seq2000 sequencing pl atform,and the sequencing results were analyzed.For high throughput sequencing,the original transcriptome data(raw reads)obtained after a series of processing were as follows: normal leaf(NL),ste m(NS),rhizome(NR)and flower(NF)of Atractylodes lanceolata were 156,096,312,127,897,750,159,229,388,145,728,860,respective ly;leaf(FL),stem(FS),rhizome(FR),and flower(NF)of Atractyl odes lanceolata var.Flower positions(FF)were 146,651,904,150,524,320,149,608,938,142,479,918 respectively.Blastx,blastp,SwissProt,TrEMBL and KEGG are used to compare and annotate all unigene s.75.99% of them are classified into one or more GO items by ge ne ontology enrichment analysis,40.51% are annotated to KO datab ase and 30.63% are annotated to KOG database.The analysis of ge ne difference between Atractylodes lanceolata and Atractylodes lance olata showed that the number of differentially expressed genes(DE G)of FS relative to NS was 298.The main pathways affecting the formation mechanism of flat stem of Atractylodes lanceolata were ko04075(plant hormone pathway)and ko04626(plant pathogen inte raction pathway),respectively,11 and 6 differentially expressed gen es.The transcriptome results were verified by qPCR in the later sta ge,and it was found that the transcriptome results were consistent with the qPCR results,which the four with high expression in FS samples were also the highest in the qPCR results.From the above results,it can be seen that phytoplasma causes changes in gene exp ression of host plants,and then causes such phenomena as flat ste m,leaf disorder,and flower leaf-changing.To sum up,this study clarified for the first time that the patho gen causing the variation of rhizoma atractylodis rhizome was phyto plasma 16sri-b subgroup,and discussed the effect of phytoplasma in vasion on the yield and quality of rhizoma atractylodis rhizoma,and further explored the molecular mechanism leading to the variation of rhizoma atractylodis rhizoma from the perspective of gene expr ession.The results of this study will provide a basis for the preventi on and control of phytoplasma diseases,and provide a basis for cla rifying the interaction between phytoplasma and host plants.