Exogenous Plant Hormones Induced Transcriptional Study Of Achyranthes Bidentata

Achyranthes bidentata Blume.is one of the famous “Four Huaiqing Chinese Medicines” and is also an important kind of traditional Chinese bulk herbs.In this study,the root and leaves of Achyranthes bidentata treated with 2 mg/L NAA + 1 mg/L 6-BA were used as experimental materials.In this research we have explored the molecular mechanism of exogenous phytohormone treatment for root accumulation of triterpene saponins.For this reason using real-time fluorescent quantitative PCR technology and transcriptome sequencing,the molecular mechanisms of key enzyme genes in the synthetic pathway of triterpenoid saponins from A.bidentata and their temporal and spatial expression specificity were evaluated.The aim is to provide a theoretical basis for the further study on cloning and functional verification of key enzyme genes in the synthesis of triterpene saponins,and the regulation of exogenous phytohormones on A.bidentata growth and the metabolism of triterpene saponins.The main results are as follows:1.For the first time,the transcriptome sequencing basic information in response to hormone-related genes in different tissues of A.bidentata was obtained.We obtained a total of 312,151,164 raw reads,of which Ab-L-CK1,Ab-L-CK2,Ab-R-CK1,Ab-R-CK2,Ab-L-T1,Ab-L-T2,Ab-R-T1,and Ab-R-T2,had 35,359,331,27,630,495,32,028,032,33,307,916,34,923,635,33,637,422,48,223,422 and 67,040,748 raw reads,respectively.After filtering low-quality reads,a total of 26.5 G of data was obtained.A total of 100,987 unigenes were obtained after assembly,with an average sequence length of 1146.8 bp,a maximum length of 15,803 bp,and a minimum of 224 bp.There are 60,356 unigenes sequences lengths between 200 and 1000 bp;23,230 between 1000 and 2000 bp;and 17,401 over 2000 bp.The number of unigenes in the short sequence was more than that of in the long sequence,which is in accordance with the general rule of the length distribution of the transcriptome sequence.2.Got unigenes functional annotation information in multiple databases.The Swiss-Prot,GO,COG,and KEGG databases were annotated with 31,634,20,488,21,715,and 12,762 unigenes,respectively 31.33%,20.29%,21.50%,and 12.64%.3.Obtained GO,COG and KEGG functional classification information of unigenes.The 20,488 unigenes classified into the 3 functional categories,molecular function,cellular components,and biological processes of the GO functional classification involved 42 and more specific subclasses.The maximum number of unigenes involved in the metabolic subclass was 14,053(68.59%).A total of 21,715 unigenes were annotated into 25 COG classifications,with a maximum of 12,007(55.29%)of the unigenes involved in the signal transduction mechanism.12,762 unigene were annotated to 303 metabolic pathways,and 377 unigenes were involved in the metabolism of terpenes and polyketides.4.Has been clear about the basic information of differentially expressed genes.20,896,9,812,28,339 and 2,731 differentially expressed genes were obtained between L-CK and L-T,R-CK and R-T,L-CK and R-CK,and L-T and R-T,respectively.There were 47 and 35 functional classes with significant difference in GO between L-CK and L-T,and R-CK and R-T,respectively.Among them,six functional sub-categories of L-CK and L-T have more functional items than R-CK and R-T.KEGG has been significantly enriched in the synthesis and photosynthesis-related metabolic pathways of terpenoids.In the treated leaves,the genes related to triterpenoid saponin synthesis were significantly up-regulated,while there was little change in the treated roots.In addition,after exogenous hormone spraying,hormone synthesis and signal transduction related genes and transcription factors in plants are also involved in regulating the synthesis of secondary metabolism triterpene saponins.5.Using KEGG metabolic pathway,395 candidate genes involved in triterpene saponin biosynthesis were excavated,including 22 MVA pathway key enzyme genes,23 MEP pathway key enzyme genes,19 triterpenoid saponin carbon skeleton formation key enzyme genes,5 oxysqualene cyclase genes,186 CYP450 genes,122 UDP-glycosyltransferase genes,and 18 glycosyltransferase genes.Using bioinformatics technology,it was speculated that UN082587 has the biological function of catalyzing the synthesis of oleanolic acid from ?-amyrin.6.The temporal and spatial expression patterns of 10 key enzyme genes in the synthesis of triterpenoid saponins were investigated by fluorescence quantitative PCR.The results showed that in addition to the higher expression levels of GGPPS and CAS genes in leaves,HMGS,HMGR,SS,SE,?-AS and CYP450 genes were highly expressed in roots and fruits.The A.bidentata transcript in response to exogenous phytohormones enriched the gene expression profile of A.bidentata,and explored a large number of candidate differentially expressed genes and candidate genes related to the synthesis of triterpene saponins.It laid the foundation for the molecular mechanism of exogenous phytohormones regulating the accumulation of triterpene saponins and the selection of a new species of A.bidentata with high triterpenoid saponin content.