Research On The Transcriptome Analysis Of Dark Green Mutant And Wild-type Plant Of Anthurium Andraeanum 'Sonate' And The Cause Of Mutation

Anthurium andraeanum Lind.is one of evergreen herbaceous perennial plant belonging to the family of Araceae.Because of its high ornamental value,anthurium has become a very popular potted flower and cut flower all over the world.A leaf color mutant has been abtained through in vitro culture of A.andraeanum 'Sonate'.Compared to the wild-type plant,the dark green mutant has darker leaf color,and the spathe color changes from pink to red too.Moreover,the anthocyanin content of leaf,petiole and spathe has significant differences.Thus the wild-type plant and the dark green mutant of A.andraeanum 'Sonate' are ideal materials for the research of anthocyanin metabolism in anthurium Anthocyanins are important secondary metabolites of plants.The metabolism of anthocyanins is regulated by many structural genes and transcription factors.In order to analyze anthocyanin metabolism through molecular level,the wild-type plant and the dark green mutant of A.andraeanum 'Sonate' were used for the research.Using transcriptome sequencing technique,differentially expressed genes related to anthocyanin metabolism and regulation can be found.Through the cloning,sequence analysis and transgenic method of these differentially expressed genes,the reason of leaf color mutation and anthocyanin content differences can be revealed.Major results are as follows:1.Determination of anthocyanin content and transcriptome sequencing were performed between the wild-type plant and the dark green mutant of A.andraeanum'Sonate'.Through the whole developmental stages,anthocyanin content of the dark green mutant were higher than the wild-type plant,and had significant difference in its early stages.Transcriptome sequencing produced 68179 unigenes and 24035 unigenes had functional annotation by comparing with various databases.Genes related to anthocyanin metabolism,transport and regulation were analyzed.All genes involved in anthocyanin biosynthesis were found,revealing the anthocyanin biosynthesis pathway is complete in A.andraeanum 'Sonate'.Genes related to anthocyanin transport and its regulation were also found.2.Based on the transcriptome data,differentially expressed genes(DEG)analysis had been performed.2161 DEGs had been filtered.Compared to the wild-type plant,1085 DEGs were up-regulated and 1076 DEGs were down-regulated in the dark green mutant.1253 DEGs were annotated.Among all DEGs,11 genes related to anthocyanin biosynthesis,3 MYB and 3 bHLH were significantly up-regulated in the dark green mutant.Through analysis,F3'H,bHLH35 and bHLH51 may be the key genes causing the leaf color mutation and anthocyanin content differences in the wild-type plant and the dark green mutant of A.andraeanum 'Sonate'.3.Based on coding sequence(CDS)of the predicted unigene from the transcriptome data,three differentially expressed genes,F3'H,bHLH35 and bHLH51 were cloned.Sequence analysis,expression vector construction and Agrobacterium-mediated transformation were also performed.Full-length CDS sequence of AaF3'H,AabHLH35 and AabHLH51 are 1536 bp,765 bp and 774 bp,encoding 512,255,258 amino acids respectively.Conserved domain analysis found that bHLH domain and ACT domain existed in both AabHLH35 and AabHLH51 gene,confirming that AabHLH35 and AabHLH51 are bHLH transcription factors and can form MBW complexes with MYB transcription factors.4.AaF3'H and AabHLH35 genes were further transformed into Arabidopsis thaliana.The seed coat color in T1 generation of AabHLH35 transgenic A.thaliana was darker than non-transgentic and the color of the leaf petiole and the base of the inflorescence axis in T2 generation were also turned purple.But AaF3'H transgenic lines had no obvious differences in phenotype.Anthocyanin content of AabHLH35 transgenic lines was 3 times significantly higher than non-transgentic plants and the relative expression level of genes involved in anthocyanin biosynthesis in A.thaliana was also higher.These results proved that AabHLH35 gene was related to anthocyanin metabolism and it was a positive regulatory transcription factor which could promote the accumulation of anthocyanin.Combining the fact that AabHLH35 was a significantly up-regulated DEG in the dark green mutant,the study considered that AabHLH35 gene is one of the most fundamental reasons for the leaf color mutation and anthocyanin content differences in the wild-type plant and the dark green mutant of A.andraeanum 'Sonate'.