Transcriptome Analysis Identifies Candidate Genes Controlling Forsythin,Forsythoside A Content And Embryo Development In Forsythia Suspensa

Forsythiae Fructus is the dried fruit of the Forsythia Suspensa(Thunb.)Vahl,and is a widely used traditional Chinese medicine.Forsythin and forsythoside A are the index components specified in the 2015 edition of "Chinese Pharmacopoeia",used to control the quality of F.suspensa raw herb and decoction pieces.At present,most of the F.suspensa in the market is wild,the content of forsythin and forsythoside A is low,and the problem of low embryo rate also exists,resulting in the inability to guarantee the quality of the medicinal materials.Cultivating high-quality varieties is the best way to solve the quality problem of forsythia.So far,the breeding of F.suspensa has not been carried out,and there is little research on the molecular breeding of F.suspensa.In order to fill the gap in the field of forsythia breeding,this study in five different periods of F.suspensa development,using RNA-seq technology without reference genome,transcriptome sequencing of F.suspensa fruit in each period was carried out,and through the biotechnology platform,the differentially expressed genes related to the content of forsythin and forsythoside A and embryo development were screened to lay the foundation for the cultivation of high-quality F.suspensa varieties in the future.The research methods and results of this subject are as follows:1.HPLC method was used to determine the content of forsythin and forsythoside A in five different periods of forsythia from June 20 to July 30.The results showed that with the growth and development of forsythia in these five periods,the content of forsythin continued to decrease,and the content of forsythoside A continued to decrease and rose slightly on July 30.2.Extract the total RNA of F.suspensa fruit samples in the above development stages for RNA-Seq sequencing,and the samples in the five stages have obtained high quality clean reads greater than 20M;obtained 161,668 unigenes from de novo assembly,and compared them with Nr,Swiss-Prot,KEGG and KOG databases to annotate gene functions,a total of 93,995 Unigenes were annotated;reference gene sequences were provided for analysis of forsythin and forsythoside A content and embryo development related genes.3.Through gene function annotation and differential expression analysis,a total of 47 phenylpropane pathway genes were found,which may encode a total of 9 enzymes in the phenylpropane pathway PAL,C4 H,4CL,CCR,HCT,C3 H,CAD,COMT,CCo AOMT;through functional annotation and phylogenetic analysis,1 DIR(DIR17),2 G4-OMT and 1 UGT were found.These genes may be involved in the biosynthesis process of forsythin,and these genes all follow the development of F.suspensa Down-regulated;related genes of forsythoside A biosynthesis pathway were found by differential expression analysis,including 2 Ty DC,6 PPO,4 PAO,1 ADH,6 UGT,1 AT and 2 RT,these The genes may be involved in the biosynthesis process of forsythoside A,and the change trend of its expression level is almost the same as that of forsythoside A content.4.A total of 14 Glus were identified through gene function annotation and differential expression analysis,these genes may be involved in the hydrolysis process of glucosidic bonds in the degradation pathway of forsythin and forsythoside A;through gene differential expression analysis and phylogenetic analysis,3 AMAs and 11 CPRs were found,These genes may be involved in the hydrolysis of rhamnosyl groups in the degradation pathway of forsythoside A and the reduction of the benzene ring,and the gene expression level is opposite to the change trend of forsythoside A content.5.Microscopic observation of the developmental morphology of F.suspensa embryos at various stages using stereo microscope,The results showed that on June 30,F.suspensa embryos began to form and gradually became larger with the development time;phylogenetic analysis and cluster analysis identified 40 candidate genes related to embryo development,including 28 PPR,1 AOS,6 WOX and 5 MADS-box,these genes may be involved in the development of F.suspensa embryos,and continue to expressed from June 20 to July 30.