Cloning And Preliminary Functional Analysis Of LdMYB6 Gene From Lilium Davidii Var. Unicolor

Lily is a perennial herbaceous bulbous flower of Liliaceae.Lily not only has high ornamental value,but also is a medicinal and edible homologous plant.The biosynthesis of anthocyanins in lily bulbs is regulated by light.At present,most of the studies on lily pigments are concentrated in flowers,but rarely in bulbs.Because the color of lily bulb affects the quality and value of commercial lily,it is necessary to study the regulation mechanism of the color change of lily bulb.In this study,the R2R3-MYB gene LdMYB6 was obtained based on the transcriptome data of Lilium davidii var.unicolor.The bioinformatics analysis,subcellular localization,protein stability analysis and tissue-specific analysis were performed.The mechanism of light treatment on the synthesis of anthocyanin in lily bulb was preliminarily investigated.The phenotypic changes of LdMYB6 gene were analyzed by overexpression LdMYB6 gene in Nicotiana benthamiana.The aim was to clarify the gene function of R2R3-MYB transcription factor LdMYB6 and provide theoretical basis for further elucidating the molecular mechanism of anthocyanin biosynthesis in lily bulbs,so as to provide effective countermeasures and technical support for the development and storage of commercial lily bulb.The results are as follows:1.In this study,transcriptome sequencing analysis of Lilium davidii var.unicolor bulbs under different illumination treatments.A total of 1,437,795,168?1.44g?raw reads were obtained,and 333,974 unigenes were obtained after assembly and splicing.Among them,16,986unigenes were annotated in seven databases,186,606 unigenes were annotated in at least one database,and 2913 unigenes were homologous with known genes of lilium species in NR database.Analysis of the differentially expressed genes between the samples,and a total of 2231differentially expressed genes were obtained.Compared with dark treatment,1207 unigenes were up-regulated and 1024 unigenes were down-regulated in light treatment.A novel R2R3-MYB gene MYB6 related to anthocyanin biosynthesis and five other key genes related to anthocyanin biosynthesis,DFR,F3H,PAL,CHS and bHLH,were screened out from the differential genes,and the relative expression of the genes under light was higher than that under dark.2.A R2R3-MYB gene LdMYB6 was obtained from Lilium davidii var.unicolor.The full-length cDNA of LdMYB6 is 705 bp,encoding 234 amino acid.LdMYB6 protein was highly conserved at N-terminal,containing R2,R3 conserved domain and conserved motif[D/E]PX₂[R/K]X₃LX₆LX₃R interacting with bHLH protein,in which the leucine?Leu?in the conserved motif interacting with bHLH protein was replaced by proline?Pro?.The characteristic conserved motif[K/R]P[Q/R]P[Q/R]P[Q/R]of AN2 subgroup was found at C-terminal.LdMYB6 was a R2R3-MYB transcription factor regulating anthocyanin biosynthesis and belonged to AN2 subgroup.3.LdMYB6 protein is located in the nucleus,which is stable under light and unstable under dark conditions,and is an unstable protein.4.The expression of LdMYB6 gene was tissue-specific,predominantly expressed in flowers and bulbs,and low in roots and stems.5.The gene expression pattern of LdMYB6 was similar to that of anthocyanin accumulation under different culture conditions of lily bulbs.The content of anthocyanin and gene expression level under light were higher than those under dark,indicating that LdMYB6 was a key transcription factor in the synthesis of anthocyanin in lily bulbs and was regulated by light.The expression level of genes related to anthocyanin biosynthesis in lily bulb development was analyzed.The LdMYB6 gene responds to the light signal and promotes the biosynthesis of anthocyanins by up-regulating the expression levels of LdPAL,LdDFR and LdCHS,which are related to anthocyanin synthesis.6.The plant expression vector pYBA1132-LdMYB6 was constructed and transformed into Nicotiana benthamiana,sixteen transgenic positive plants were obtained.Compared with wild type,the flowering period of transgenic tobacco was 3-4 days earlier and the leaves were obviously larger.