Isolation And Analysis Of AGAMOUS Homologous Gene And Its Promoter From Vicia Sativa L.

Common vetch (Vicia sativa L.) is a good forage cultivateded in alpine steppe, which could provide spring proteide feed for stocking in such areas. However, little has been known about the molecular mechanism of floral organ development process of this species. It restricted the application of molecular method on this species. AGAMOUS(AG) gene is one of the most important floral homologous genes involved in plant stamen and gynoecium development. In this study, common vetch VsAG gene was isolated from breeding line 2505 using hiTAIL-PCR, RACE and RT-PCR, and analysed using bioinformatics method. The major results are as following:(1) An AG homologue gene and its upstream regulation sequence were isolated from common vetch. This gene was 3158 bp in DNA sequence, coded a protein product of 244 animo acides. Homological analysis showed that the similarity between this sequence and PsAG factor of Pisum sativum was over 98%. Therefore, the sequence was considered to be common vetch AG gene. It was named VsAG with the accession number JF313850 in GenBank. Based analysing the cis-elements existing in upstream promoter region and the second intron, it is considered that VsAG second intron region sharing some similar cis-elements with promoter region. And a binding site of LFY factor and some positive elements were also found in this region. It indicated that the second intron of VsAG gene could play an important role in temporal and spacial regulation of VsAG expression.(2) The role of VsAG in evolution was determined through phylogenetic analysis based on several AG family menbers of angiosperm plants. It was assumed that euAG class and PLE class were created in the evolution of AG family while VsAG belongs to euAG class. The results also indicated the duplication of VsAG.(3) To provide internal standard for expression analysis of VsAG and other genes via qPCR, partial sequences of actin gene VsACT7(HM004434), VsACT11 (GU946218) and glyceraldehyde-3-phosphate dehydrogenase gene VsGAPDH(HM 117006) were isolated from common vetch. Thess genes also enriched materials for molecular breeding.