Clone Of The NtSAMT Gene In Narcissus Tazetta L.Var. Chinensis Roem And Transformation Of Asarina Procumbens

Narcissus tazetta var. Chinensis Roem is a bulbous perennial flower belonging to Amaryllidaceae, flowering in winter, it is one of the ten famous flowers in China. The colour of its petals are elegant and rich of aroma. Asarina procumbens is a perennial crawl vine belonging to Scrophulariaceae with numerous leaves and elegant color, which is good ornamental plant for vertical greening in the garden. However, it lacks the fragrance and reduces the ornamental value.Fragrance is a kind of secondary metabolites, consists of a large number of volatile substances, in the form of volatile spread into the environment. The role of fragrance mainly can promote plants of ornamental value, on the other hand can attract insects pollination, through hybridization breeding new varieties. At present, the main research is terpene compounds of metabolic pathway and styrene acrylic acid type compounds/benzene compounds of metabolic pathway. Research has shown that by introducing exogenous floral material synthesis related genes, make transgenic plants flower quality change is feasible. Recent studies show that SAMT genes are important catalytic enzyme phenylalanine metabolic pathway, in the metabolic regulation of floral products play an important role. This study uses the means of molecular genetics, cloning of flowers synthesis related genes in Chinese narcissus Nt SAMT, analysis of the structure and function of the gene. Turn the Nt SAMT gene by agrobacterium mediated method to the Asarina procumbens, to improve the Asarina procumbens value; At the same time for ornamental flowers provide a scientific basis for genetic improvement.In this study, with the flowering petals of Narcissus tazetta var. Chinensis Roem as materials,Nt SAMT was cloned by RT-PCR. By using bioinformatics, the structure of the coding protein was predicted, and the multiple sequence alignment and phylogenetic tree were analyzed. A plant expression vectors based on pBI121 with CaMV35 s promoter were constructed. Genetic transformation system of Asarina procumbens were established. The Positive transgenic plants were obtained by resistance screening PCR and RT-PCR analysis.The main results were summerized as following:(1) By RT-PCR, Nt SAMT gene was cloned, which had a total length of 1269 bp with an open reading frame of 1131 bp, encoding 376 amino acids. The number of amino acids is same with Narcissus tazetta which had logined in GenBank as No.AFR23078, the nomologous of them is99.2%. There are 3 amino acids residue differences between them, respectively is H20 and Y20,L62 and S62, L143 and F143.(2) The hydrophilic and hydrophobic analysis of the protein, the relative molecular weight ofNt SAMT gene was 42.46 kD and the isoelectric point was 5.11. Compared the amino acid sequence of NtSAMT and other plant, multiple sequence alignment analysis indicated that the homology is between 41.6% and 47.2%. Molecular phylogenetic tree showed that the Nt SAMT cloned gene Nt SAMT with the China narcissus SAMT gene(registration number for AFR23078) had logged-in GenBank have a high homology and belonging to the same cluster. However, compared with other containing the gene plants have a low homology.(3) Genetic transformation system of Asarina procumbens had been established. Small scales as transgenic receptor, pre-culture 10 days; Infection 15 minutes with bacteria of OD600 value of about 0.5~0.6; Co-culture with medium for 3 days; The original filter medium is MS + 1. 5 mg/L6-BA + 0. 3 mg/L NAA + 0. 3 mg/L KT + 20 mg/L Km + 350 mg/L Cef. The best transgenerational screening culture medium is MS + 2. 0 mg/L 6-BA + 0. 2 mg/L NAA + 60 mg/L Km + 350 mg/L Cef. The best induced root filter medium is MS+150 mg/L Km(4) The Nt SAMT gene into Asarina procumbens by Agrobacterium-mediated method,acquired resistance plant 42. There were 25 plants transformed Nt SAMT had been tested positive by PCR, PCR positive rate was 54.8%. Randomly selected eight strain positive plants for further detected the positive plants above by RT-PCR, the results of plants transformed NtSAMT shows that 5 plants RNA had transcribed expression. Prove that the purpose gene can transcription normally in transgenic plants.