Cloning And Sequence Analysis Of AP3-3 Gene In Normal Plant And Natural Variant From Anemone Rivularis Var. Flore-minore

Flower is the unique organ for angiosperms which plays important functions in terms of reproduction and appreciation. The floral development is a very important physiological process that affects the survival and reproduction of angiosperms. Through the research on reasons and mutation mechanism of variant plants, it is helpful to further understand the evolutionary relationship of floral organ development along with explain and modify molecular models of flower development, and has great application value and significance for the development of research on flower type directional breeding.The study takes normal plant and tepal natural variant which have big differences in appearance as materials from wild Anemone rivularis var. flore-minore, class B genes were isolated from genomes of normal plant and natural variant by using conventional and Hi-Tail PCR techniques. After that, carrying out the comprehensive comparative analysis of the two?s sequence characteristics and predicted proteins using the bioinformatics knowledge, in order to explore the possible reasons for the variation of tepals in variant plants. The following main results were obtained by analysis:(1) The two class B genes which isolated from genomes of normal plant and natural variant were all proved to be AP3-3 branch of paralogs which belong to AP3 gene family from B class MADS-box genes, and named NArAP3-3(in normal plant) and VArAP3-3(in natural variant) respectively.(2) The full length of NArAP3-3 gene was 3795 bp whereas 3898 bp in VArAP3-3, they both have a 666 bp coding sequence(CDS) and can encode 221 amino acids. The protein showed a typical MADS-box gene structure containing MADS domain, K domain, I region and C terminus. The comparison result of full length sequences between NArAP3-3 and VArAP3-3 genes was found to have a 49 bp insertion in VArAP3-3 gene, and there are also 4 base mutations in ORF sequence compared with the NArAP3-3 gene.(3) By bioinformatics analysis of the two’s full length sequences, 221 amino acids and insertion sequence, we can find that there are differences in all aspects of gene promoter, basic properties of protein, structure function domain, advanced prediction structure and so on.These differences may be one of the causes of tepals variation, the changes of tepals and stamens are likely to have a certain correlation with the VArAP3-3 gene in variant plants of Anemone rivularis var. flore-minore. The changes of the regulatory region and coding sequence in VArAP3-3 gene may cause the increase of expression level and the mutation in structural properties of the target protein that make the stamens degenerated and reduced, the degenerated stamens may turn into tepals to increase its number and the number of rounds at the same time. This study lays the foundation for the further exploration of its variation mechanism.