PeNAC67 Gene Cloning And Molecular Mechanisms In Development Of Lip In Phalaenopsis Equestris

Phalaenopsis is a member of the Orchidaceae.It is native to tropical rain forest areas with fleshy roots and epiphytic growth.The flower color is magnificent and the flower shape is peculiar: 3 petalized sepals,2 lateral petals and 1 lip;forming a bilaterally symmetrical structure.The unique color and flower shape of the lip attracts pollinators and provides a platform for pollinators to collect nectar.Typical "ABCDE" model and "quartet model" can not explain the developmental mechanisms of floral organ development in Phalaenopsis.The research on floral organ development mainly focuses on the MADS-box gene,and the upstream substitution factor or interaction protein of the MADS-box gene has become new research direction.The orchid model plant is Phalaenopsis equestris(P.equestris)and its genome was announced in 2015.Natural mutants(the side petals mutate into a lip-like structure,called peloric mutant)as researching materials for revealing the development of the lip.We discovered PeNAC67,a key transcription factor major in regulating the development of lip,and used Assay for Targeting Accessible-Chromatin with highthroughout sequencing technology.The full length of PeNAC67 is 1356 bp,the open reading frame is 981 bp,and it encodes 326 amino acids.It is a transcription factor of the NAC protein family.PeNAC67 has typical NAM domain,including five subdomains A,B,C,D,and E.In addition,PeNAC67 is related to Dendrobium catenatum and Apostasia shenzhenica clusters are close and belong to the monocotyledonous branch.PeNAC67 is located in the nucleus and has transcriptional activation activity.The C-terminal amino acid plays an activating activity.According to q RT-PCR analysis,PeNAC67 is ubiquitously expressed,but the expression level in reproductive organs is significantly higher than that in vegetative organs,and the expression level increases significantly with the development of floral organs.In tobacco,the virus induces Nb NAC18 gene silencing and the number of petals increases.However,in P.equestris,virus-induced silencing of the PeNAC67 gene caused the Liplike petals to restore the wild-type petal phenotype,indicating that PeNAC67 positively regulates the development of the lips.Furthermore,the silencing PeNAC67 in "Big chill" shows that the distance between the petals becomes smaller.In summary,it can be seen that PeNAC67 is closely related to the development of floral organs,and the PeNAC67 is directly regulating the development of lips.It is known that the MADS-box gene of B-class plays an important role in floral development.Yeast one-hybrid experiments have found that PeNAC67 is not upstream of the MADS-box gene.However,the two-hybrid and Bi FC assay indicate that PeNAC67 interact with PeMADS3.q RT-PCR analysis of the expression levels of the vital MADS-box genes in the "P code" showed that the expression of MADS2 and MADS10,which are closely related to petals,was significantly higher than that of MADS3 and MADS9,which are related to lip.In summary,this study reveals the molecular mechanism of orchid lip development from two levels of interaction and coexpression mechanism,which will lay the foundation for orchid research.