The Functional Study Of DoMYB5 And DoMYB92 Transcription Factors In Dendrobium Officinale

Dendrobium officinale,a perennial herb of the Orchidaceae family from the genus Dendrobium,is a valuable ornamental and medicinal plant that has a long history of cultivation in China and is highly valued for its attractive blossoms and excellent nutritional content.Dendrobium officinale ’s red organ perfectly integrates people’s demand for beautiful horticultural and therapeutic plants.It has gotten increasingly popular and more expensive since entering the consume market.Anthocyanins have been demonstrated to influence the production of red organs in Dendrobium officinale,with R2R3-MYB transcription factors playing a critical role in anthocyanin biosynthesis.This study aimed to investigate these transcription factors in two Dendrobium officinale species,validate their functions,and explored some of their regulatory mechanisms in the anthocyanin biosynthesis pathway.The primary findings are summarized below:1.Two R2R3-MYB genes,Do MYB5 and Do MYB92,were obtained from Dendrobium officinale using RT-PCR.Bioinformatics analysis suggested that both Do MYB5 and Do MYB92 possess two conserved SANT domains.Do MYB5 is closely related to Dh MYB2 in Dendrobium hybrid,whereas Do MYB92 is closely related to Do MYB105 and Arabidopsis thaliana’s At MYB4.Do MYB5 exhibited higher expression levels in the red organs of Dendrobium officinale,while the expression of Do MYB92 was lower in the same.2.The expression vectors p TRV2e-Do MYB5 and p TRV2 eDob HLH24 were constructed consequently.Agrobacterium Tumefaciens was infused into the green leaves of Dendrobium officinale leading to the appearance of red patches in the injection area of the leaves.Furthermore,it was observed that the injection of Do MYB5 and Dob HLH24 promoted a substantial increase in the anthocyanin content in the leaves of mixed injection when compared to single injection.Thus,it is evident that the transcription factors Do MYB5 and Dob HLH24 can effectively enhance anthocyanin accumulation in Dendrobium officinale leaves,as their effects are particularly noticeable when acting conjunction.3.Tobacco plants were genetically transformed using the leaf disk method to produce transgenic plants containing Do MYB5 and Do MYB92 genes.Our research also introduced Do MYB105 and Dob HLH24 genes into tobacco to obtain transgenic plants.The OE-Do MYB5 transgenic plants showed a significantly change in the colors of the flowers,with a deepening of the color of corolla,calyx,anther,filaments and ovary,while the OE-Dob HLH24 transgenic plants showed a change only in the color of corolla.The corolla colors of the OE-Do MYB92 and OE-Do MYB105 transgenic plants were lighter.Fluorescence quantitative PCR analysis of the flower crown of transgenic tobacco revealed that Do MYB5 and Dob HLH24 promoted the expression of structural genes in the anthocyanin biosynthesis pathway of transgenic tobacco.Among them,Do MYB5 led to a significant upregulation of Nt CHS and Nt DFR,and Dob HLH24 promoted the expression of Nt UFGT the most.Conversely,both Do MYB92 and Do MYB105 inhibited the expression of Nt CHS,Nt DFR,Nt ANS and Nt UFGT.The results indicated that the anthocyanin,proanthocyanidin,and total phenol contents of OE-Do MYB5 and OE-Dob HLH24 plants were significantly enhanced,while those of OE-Do MYB92 and OE-Do MYB105 plants were dramatically reduced.In summary,our results demonstrate that Do MYB5 and Dob HLH24 positively regulate anthocyanin biosynthesis in tobacco plants,whereas Do MYB92 and Do MYB105 negatively regulate it.4.Through yeast two-hybrid and double luciferase complement(Luc)tests,it was discovered that the transcription factors Do MYB5 and Do MYB92 can interact with the transcription factor Dob HLH24 to form heterodimers.The dual luciferase reporter assay system analysis revealed that the injection of Do MYB5 and Dob HLH24 alone could activated the Do CHS and Do DFR promoters of Dendrobium officinale,while the combined injection of the two had a more greater activation effect on Do CHS and Do DFR promoter activities.On the other hand,Do MYB92 decreased the activity of Do CHS and Do DFR promoters.The results of the yeast single-hybridization experiment showed that Do MYB5 and Do MYB92 interacted with promoters of Do CHS and Do DFR,respectively.In conclusion,Do MYB5 positively regulates anthocyanin biosynthesis in Dendrobium officinale by regulating the promoters of structural genes Do CHS and Do DFR,while Do MYB92 negatively regulates anthocyanin biosynthesis pathway in Dendrobium officinale by regulating the promoters of structural genes Do CHS and Do DFR.