Characteristics Of Anthocyanin Biosynthesis Pathway In Spathiphyllum Kochii ’Mojo’ And Functional Analysis Of Related Genes

Spathiphyllum kochii is one of evergreen herbaceous perennial plants,which belongs to the family of Araceae.Because of its high ornamental value,S.kochii has become a very popular potted plant and a cut flower all over the world.However,due to the limited number of varieties in S.kochii,the industry is restricted.It is important to enrich the species resources of S.kochii.Anthocyanins are important secondary metabolites of plants.Anthocyanins regulate the color of flowers and fruits of plants.Anthocyanins biosynthesis is regulated by many structural genes and transcription factors.In order to analyze anthocyanin biosynthesis at molecular level,S.kochhi ’Mojo’ was used in the research.Using transcriptome sequencing technique,key genes related to anthocyanin biosynthesis have been identified.Through the cloning,sequence analysis,transient expression assay and yeast two-hybrid analysis of these key genes,the reason of lacking anthocyanins in spathe is revealed.The results are as follows:1.Mature spathes of spathiphyllum were used for histological observation by free-hand section method.The transverse section observed under microscope indicated that chlorophylls and carotenoids were normally synthesized and there was no anthocyanin in spathe cells.Transcriptome sequencing of S.kochii ’Mojo’ spathe was further conducted to analyze the anthocyanin biosynthesis pathway.Transcriptome sequencing produced 79947 unigenes and 28188 unigenes had functional annotation by comparing with various databases.With qRT-PCR results,genes related to anthocyanin biosynthesis were analyzed.The results showed that the anthocyanin biobiosynthesis pathway was intact in spathiphyllum.However,the expression of Anthocyanidin Synthase(ANS),the pupative key gene was extreme low.Transciption factors related to anthocyanin regulation were also found.Among these,MYB27 encoding a MYB transcription factor,MYB27 is probably the key transcription factors for anthocyanin biosynthesis.2.According to the coding sequence(CDS)of the predicted unigene from the transcriptome data and NCBI data,three key genes,ANS,MYB27,bHLH were cloned.Full-length CDS sequences of SkANS,SkMYB27 and SkbHLH are 1092 bp,669 bp and 1218 bp,encoding 364,223,406 amino acids respectively.Conserved domain analysis found that MYB domain existed in SkMYB27 gene and bHLH domain existed in SkbHLH gene,confirming that SkMYB27 is MYB transcription factor,SkbHLH is bHLH transcription factor.3.Using transient expression assay,SkMYB27 was overexpressed in Anthurium andraeanum.The color of A.andraeanum-spathe became pale color,and.the anthocyanins content of spathe was reduced significantly.Yeast two-hybrid assay was carried out to test protein interactions between SkMYB27 and SkbHLH,the result showed that SkMYB27 could interact with SkbHLH.