Studies On Transferring MT/αα Gene Of Mice Into Aquatic Canna

Canna has strong vitality and widespread ecological amplitude, and it can adapt to all kinds of climates in our country, which had been cultivated ranging from the tropical, subtropical to temperate regions. Mammalian metallothioneins (MTs) are a sort of low-molecular-weight, cysteine-rich and metal-binding proteins, which mainly participated these processes such as the metabolism of essential metal ions and the detoxification of toxic heavy metals in animals. Mice's MT-â… hasαandβtwo domains, which are not only interrelated but also independed on each other in the structure and function. They have different affinity toward the heavy metals,α-domain binding Cd²⁺, Hg²⁺ preferentially andβ-domain favoring to Cu²⁺, Zn²⁺.In this study, the author used aquatic Canna as experimental material and initiated the research of the genetic transformation regarding mouse metallothionein (MT) gene and its mutant geneααby Agrobacterium-mediated transformation. The research aims at utilizing the MT's characteristics of the conjunction and detoxification of heavy metals to increase Canna's resistance and accumulation toward heavy metals, such as Cd, thus developing a new type of plant, which is used for the treatment of heavy metal pollution in the environment.Preparation of aseptic seedling: the wild aquatic Canna was used as the materials of this experiment. They were rinsed with flowing water about 20 minutes after ablution, then cut the buds 1-2 cm long, immersed into 75% alcohol about 30 seconds under the sterile environment, put into 0.1%HgCl2 about 10 minutes, In the end, cut about 5mm3 stem apex with 3-4 leaf primordium as explants, and inoculated in proper nutrient medium after emundationing 4-6 times with SW.Tissue culture: aquatic Canna was used as experimental material. In the study of the induction conditions of multiple buds and callus, different explants of aquatic Canna were inoculated into different media which were added into different types and concentration of exogenous phytohormone. The results showed that: The stem apex growing point and pedicel were appropriate organ to tissue culture; The phytohormone concentration which promote the highest survival rate of seedling was NAA 0.1mg/L and 6-BA 2.0mg/L or 3.0mg/L; Seedling of the highest average height growed on the medium, which phytohormone prescription was 6-BA 2.0mg/L or 3.0mg/L and NAA0.5mg/L; The optimal medium of Canna adventitious buds proliferation was MS+6-BA8mg/L + NAA0.1mg/L.Determination of selective pressure of genetic transformation: The selectable marker gene of the plant expression vector were npt-â…¡and Bar gene, so kanamycin and PPT were both adopted as selective agents in genetic transformation. In this study, we had undertook a systematic study on the kanamycin resistance of Canna and found that the cotyledon albinism rate increased, the rhizome shorter, the green weight reducing with the increased concentration of Kan. In the treatment of 30 mg/L kan, seedlings' albinism rate reached 100% after 20 days. The kanamycin resistance test of Canna played the foundation for genetic transformation and screening of positive posterity.Optimization of the system of genetic transformation: Taking aquatic Canna growth point as materials, we studied several factors of influencing Agrobacterium- mediated transformation system of aquatic Canna growth point, and drew the Optimal conditions: bacterial concentration OD600=0.3-05, AS 100μmol/L, infection time 15 min, and co-culture time 3 days.The author tried to transfer MT (αα) gene into Canna by the means of Agrobacterium tumefaciens infecting Canna growing point, then resistant plantlets were selected and obtained on the selective medium of Kan. Using this method, growing point generated cespitose shoots directly and hadn't callus stage. In the whole transformation experiments, 281 growing points were infected, 25 resistant plants were obtained.