Cloning And Analysis Of AGAMOUS Homologue Gene LLAGM1 And LLAGM2 From Lilium Spp.

Lilies are one of the most beautiful flowers with pleasant scent on the world. However, no multiple corolla flower lilies appeared in China. In addition, lilies have large amount of pollen, which may cause pollen contamination during cut flower collection, selling and consuming. For this purpose of modifying the flower form of lilies, we conducted a series of researches to try to understand the expression of genes related to the flower development. In this thesis, we present two major results both on the experiment methods improvement, and Cloning and analysis of AGAMOUS homologue Gene related to the lily spp. flower development. The main results as following:1. It is extremely impartment to extract high quality total RNA from plant tissues. We compared the quality of total RNA extracting by five methods, CTAB, TRIZOL, WI, GT, and TRIS-SDS respectively. And three materials were used from Lilium sections, Oriental cutting flower on the market, Lilium lanc(?)olium and Lilium long(?)olorum our germplasm conservation field. The results indicated that, the methods CTAB, TRIZOL, WI were not suitable for the high quality extraction RNA from the lilies. But the high quality RNA can be achieved by the TRIS-SDS method, which may fit to different Lilium spp. The improved TRIS-SDS method was simple and convenient, and the cost of the experiment may be cut, for most of the reagents were regular put up in the laboratory.2. They were members of MADS box gene family, and which highly homologous to AGAMOUS from Arabidopsis and other known orthologues. LLAGM 1 and LLAGM 2, the two cDNA fragments associated with flower development were cloned from Lilium by the RT-PCR-based cloning method and degenerate PCR primers against conserved sequences in the AG homologue of several plants in this study.The LLAGM 1 cDNA fragment was around 1000bp in length. After the elimination of protein sequence the LLAGM 1 was estimated a product in 243-amino acids. It is appeared that the LLAGM1 shares 68.5% amino acid similarity with AG of Arabidopsis, 68.5% with CaMADS1 of Corylus avellana, 73% with HAG1of Hyacinthus orientalis, 92.2% with LLAG1 of Lilium long(?)lorum, 68.2%with OsMADS3 of Oryza sativa, and 71% with PeMADS1 of Phalaenopsis equestris and.71% with pMADS3 of Petunia hybrida respectively. There were eighteen amino acids difference shown in K domain and two in the C-terminal between LLAGM 1 and LLAG1.LLAGM2 cDNA fragment was around 1168bp in length. After the elimination of protein sequence the LLAGM2 was estimated a product in 249-amino acids. It is appeared that the LLAGM2 shares 64.1% amino acid similarity with AG, 64.6% with CaMADS1, 68.6% with...