Cloning And Function Analysis Of AGAMOUS Homologous Genes From Hemerocallis Spp.

Daylily(Hemerocallis spp.)is a perennial herb that has been cultivated in China for thousands of years,the complementary modification of the flower type of daylily flowers is one of the important research objectives of its breeding.According to the flower development ABC model,this study cloned AGAMOUS gene,a C class functional gene in the MADS-box family that determines stamen and petal formation,from six daylily species with different petalization degrees.Then we conducted bioinformatics analysis of its encoded protein,studied the gene expression pattern by real-time quantitative PCR,and constructed the daylily AG homolog gd Hf AG over Expression vector 1301-Ubq-AG-Flag,the overexpression vector was transferred into wild-type Arabidopsis by Agrobacterium infestation to obtain gd Hf AG overexpressing transgenic Arabidopsis.The main results of this paper were as follows.a.The Hf AG genes of the three single and three heavy flower daylilies obtained by cloning,named qh Hf AG,gd Hf AG,gdf Hf AG,dm Hf AG,dm Hf AG and sds Hf AG,respectively,had the same CDS sequence length of 623 bp and encoded 203 amino acids,but each amino acid sequence had some differences compared to the other five,in which,at position 83 of the coding amino acid sequence,both were serine(Ser)in the single flower variety and threonine(Thr)in the heavy flower variety,and the change of amino acid sequence due to base mutation might be one of the factors arising from the single flower and heavy flower varieties of daylily.b.Domain analysis revealed that the Hf AG gene has a typical plant MADS-box gene structure,with the AG gene starting from the 5’ UTR,followed by M domain,I region,the K domain,C region.The amino acid sequence of the C region is more variable but often contains some conserved motifs,but in the present experimental results,the conserved motif AGⅠ were existed and AGⅡ were lost in the Hf AG gene of the single flower variety and the heavy flower variety,so for these daylily varieties,it can be presumed that the mutation of the conserved motifs in the C region is not the cause of double flower.c.Comparative analysis with the known sequences in the NCBI database showed that the Hf AG gene had high identity with the AG homologs of several monocotyledons,which belonged to the AG genealogy,and the AG homologs of three single flower variety clustered in one branch,and the AG homologs of three heavy flower varieties clustered in another,so it was possible that the AG homologs of single and heavy flower daylilies had produced gene duplication event.d.The secondary structure and tertiary structure of the protein were predicted and analyzed.The secondary structure of Hf AG protein is dominated by α-helix,mainly concentrated in the K domain,followed by random coil,while the extended strand accounted for less,and the lowest proportion was β-turned.Overall,the Hf AG genes of both single and heavy flower daylilies had some changes in secondary structure due to amino acid changes,and the secondary structure was also greatly altered in the C region without amino acid site mutations.The tertiary structure of the two conserved regions of Hf AG protein,M domain and K domain,was later predicted by homology modeling,and the tertiary structure of the mutated sites in M domain was slightly more changed than that of K domain,but did not match the similar tertiary structure of the non-conserved I region.e.The tissue-specific expression patterns of the six obtained Hf AG genes were analyzed,and the results showed that the overall expression trends of Hf AG genes in heavy flower daylilies were relatively consistent,with higher expression in stamens and carpels,lower expression in sepals and petals,and expression in all four whorls of floral organs of the single flower varieties.That is to say,relative to the heavy flower varieties,the AG homologous genes in the single flower varieties showed an expression pattern extending from the inner whorl to the outer whorl,indicating that the gene may be involved in the developmental process of daylily petalization,and the change in expression range may lead to the formation of heavy flower daylilies.f.The overexpression vector 1301-Ubq-AG-Flag containing gd Hf AG gene was constructed,and the transgenic plants were obtained by transfecting Arabidopsis.Compared with the wild type arabidopsis thaliana,the transgenic plants showed poor growth,dwarfed plants,narrower leaves with front curls,and some leaves with yellowing.On top of the transcriptome sequencing and genome sequencing data of research group,this study cloned,bioinformatics analysis,expression pattern analysis and gene transformation of class C homologous gene Hf AG from single flower daylilies and heavy flower daylilies,to further investigate the functions played by these genes in the process of daylily flower organ formation to provide the theoretical basis,so as to provide the research of daylily flower type variation mechanism and through genetic engineering means to conduct daylily heavy flower breeding.