Full-Length Transcriptome Analysis And Exploration Of MYB Transcription Factors Related To Anthocyanidin Synthesis Of Eleutherine Bulbosa(Mill.) Urb.

Eleutherine bulbosa（Mill.）Urb.,is a traditional folk medicinal plant distributed in the southwest region of China.At present,Eleutherine bulbosa（Mill.）Urb.is of great value in the development and utilization of physiological active substances,but the research in the field of molecular biology is relatively scarce.The bulb site of Eleutherine bulbosa（Mill.）Urb.is rich in anthocyanin,and the molecular mechanism of its anthocyanin biosynthesis is of great theoretical and practical significance for the development and utilization of natural pigments in its bulbs.Based on the analysis of full-length transcriptome sequencing data,screening out MYB transcription factors involved in biosynthesis of the bulb of Eleutherine bulbosa（Mill.）Urb.,construct overexpression vector to verify gene function of the candidate transcription factors for,and using the VIGS to verify the gene function,so that we can analyze the molecular regulation of the target MYB transcription factor involved in anthocyanin biosynthesis.The main study results are as follows:1.Full-length transcriptome sequencing and analysis were performed by using the Pac Bio Sequel^TM sequencing platform,High-quality Isoforms sequences of182,873 were obtained,Functional annotation was obtained with 105,734 transcripts in the Nr database alignment;There were 90,207 transcripts being functionally annotated in the Swissport database;There were 77,965 transcripts available for the KOG functional annotation;According to the KEGG database,There were 52,047 transcripts obtaining functional annotations,A total of 132 metabolic pathways including secondary metabolites synthesis,antimicrobial synthesis,sugar and starch metabolism were obtained.A total advanced annotations of 105,976 CDS obtained,predicted a total of 3,257 transcription factors.For the structural analysis,a total of39,666 SSRs were identified,for lnc RNA without annotated to the four major databases,a total of 6,394 lnc RNA were predicted.The Full-length transcriptome sequencing of Eleutherine bulbosa provided a reference for deeply studying the function of MYB transcription factors and regulatory mechanisms.2.Based on full-length transcriptome sequencing data,we have screened out Unigene0006851（UBQ）as the reference gene of Eleutherine bulbosa（Mill.）Urb.for the fuctional research of MYB transcription factor.Using the sequence of At MYB75 transcription factors related to the biosynthesis of Arabidopsis as a reference,Eb MYB5 and Eb MYB52 were the target MYB transcription factors through the multiple sequence homology alignment and phylogenetic analysis,combined with the spatiotemporal expression levels of primary screening transcription factors in the bulbs of different developmental periods of Eleutherine bulbosa（Mill.）Urb.3.We used the c DNA sample of Eleutherine bulbosa（Mill.）Urb.as the template to obtain full-length sequences of transcription factors of Eb MYB5 and Eb MYB52 and constructed stable genetic overexpression vectors.By using heterologous genetic transformation into Arabidopsis and analyzed expression levels of overexpressed genes using q RT-PCR technology,we obtained Eb MYB5 and Eb MYB52 transgenic lines of transgenic plant.The results showed that the relative expression levels of Eb MYB5 and Eb MYB52 Arabidopsis transgenic lines were significantly higher than in the wild-type plants,and pigment accumulation occurred in the transgenic lines.At the same time,we used the VIGS system to analysis the function of Eb MYB5 and Eb MYB52 in Eleutherine bulbosa（Mill.）Urb.plant after VIGS treatment,the result showed that the relative expression level of Eb MYB5 and Eb MYB52 and gene related to the biosynthesis of anthocyanin appeard in a down regulation trend,which was an evidence to prove that Eb MYB5 and Eb MYB52 are involved in the regulation of biosynthesis of anthocyanin.