Transcriptome Analysis Revealing Flower Coloration Of Sophora Japonica Linn.and Function Analysis Of Key Genes Involved In Anthocyanin Accumulation

The pagoda tree(Sophora japonica L.)is a member of deciduous trees of Papilionaceae,which has a cultivation history over 3,000 years in China.The flower color of the pagoda tree has always only been observed to be white or light yellow-green,which greatly limits its ornamental value.Many studies have shown that the petals of the pagoda tree have abundant flavonols which is the substrate for anthocyanin production.But whether there are anthocyanins in its petals?and what is the type of the anthocyanins?and how much is the content of anthocyanins?Studies on these issues have not been reported.Here,we found a unique pink flowering mutant of the pagoda tree of which the flag,wing and keel petals are pink after many years of breeding work.However,the mechanism for the mutation of flower color in this pagoda tree is unclear.The petals of pagoda trees are rich in flavonoids such as rutin,which are the precursors for anthocyanin synthesis.Hence,we conjectured that the content or type of anthocyanin in the petals PM has changed,resulting pink coloration.In order to prove this conjecture,we used petals of different stages of pink-flowering mutant(PM)as research materials,and the petals of wild type(WT)of corresponding periods as controls.Through qualitative and quantitative analysis of anthocyanins and transcriptome sequencing analysis,we identified some key genes for pink flower formation.In addition,isolation and verification of the full coding sequences(CDS)of and key genes were also performed in this research.The main results were as follows:(1)Anthocyanins in petals of PM and WT at four stages were determined using High Performance Liquid Chromatography-Diode Array Detection-Mass Spectrometry(HPLC-DAD-MS~2)system.As a result,two kinds of anthocyanins and eight flavonols were detected in both PM petals and WT petals.The total flavonol content in the petals of PM and WT is very abundant,but there were no significant differences between WT and PM.The content of total anthocyanins in petals of PM were significantly higher than that of WT at each stage.(2)Comparative transcriptome analysis was carried out using petals of PM and WT from four floral stages.Based on GO enrichment and KEGG pathway enrichment analysis,14 anthocyanin biosynthesis structural genes,and the unigenes c150127.graph?c0 encoding DFR enzyme may play a key role in anthocyanin accumulation in PM petals.In addition,the key anthocyanin synthesis-regulated regulatory genes were screened including Sj PAP1,Sj MYB111,Sj MYB1b,Sj MYBL2,Sj TTG1,Sj TT8,Sj JAZ3,Sj ERS1,Sj PHYA,Sj CI7s,Sj HY5,Sj IAA19,Sj AXR3 and Sj IAA27.(3)The CDS of the key gene Sj DFR(c150127.graph?c0)is isolated from pagoda tree,encoding 339amino acids.Multiple sequence alignments analysis showed that the protein sequence of Sj DFR was highly homologous to those DFR proteins in other species,and had a NADPH binding site(V10?Y30)and a substrate-specific conserved motif(T131?V156).The Sj DFR had a tissue-specific expression pattern,and its relative expression levels were related to anthocyanin content.It had higher expression levels in petals with high anthocyanin content.The Sj DFR gene was introduced into tobacco via Agrobacterium-mediated transformation and the over expression of Sj DFR increased anthocyanin production in flowers of transgenic tobacco,resulting in flower colors being deepened to varying degrees.q RT-PCR analysis showed that anthocyanin content in corolla of transgenic tobacco correlates with transcription levels of Sj DFR.(4)The key transcription factor Sj PAP1 was isolated from pagoda tree.Phylogenetic analysis showed that the Sj PAP1 had closer relationship with members in subgroup 6 and subgroup 7 of the R2R3-MYB of Arabidopsis thaliana.The overexpression vectors were constructed and transformed into tobacco.The color of leaves,corollas and other tissues of these five transgenic tobacco lines shifted to red to varying degrees,and the content of anthocyanins in leaves and corolla increased significantly compared with the control.Transcription levels of anthocyanin biosynthetic genes and regulator genes Nt AN1a and Nt AN1b in the corollas and leaves of transgenic tobacco were significantly up-regulated compared to the control.