Cloning And Functional Analysis Of Methyl Halide Transferase

People have taken an interest in the natural sources and biological production mechanisms of VHC (volatileh alohydrocarbon) since we know CFCs can destroy the ozone of stratosphere. Methyl chloride (CH₃Cl) and methyl bromide (CH₃Br) are the primary carriers of natural chlorine and bromine, respectively, to the stratosphere, where they catalyze the destruction of ozone. Meanwhile CH₃Br has ever been a broad used fumigation because its virtues, but now was restricted by the Montreal Protocol due to its bad impact to the environment.Despite the economic and environmental importance of these methyl halides, their natural sources and biological production mechanisms are studied a little. Besides CH₃Br fumigation, important sources include oceans, biomass burning, tropical plants, salt marshes and certain crops and fungi. Algae and fungi produce both methyl halides and polyhalomethanes, while plants produce only methyl halides. Methyl halides are products of methyltransferase activity, the reaction is like this: SAM + X - (Cl^-, Br^-, or I^-)→CH₃X + S-adenosylhomocysteine. A survey of 118 herbaceous species, hased on CH₃I production by leaf discs supplied with KI, detected the presence of in vivo halide methyltransferase activity in 87 species and probably the halide methyltransferase is ubiquitous in higher plants. Whether or not the halide methyltransferase is related to naturally occurring salt tolerance, it may be of interest for improvement of salt tolerance via metabolic engineering.The model plant Arabidopsis thaliana produces and emits methyl halides and that the enzyme primarily responsible for the production is encoded by the HARMLESS TO OZONE LAYER (HOL) gene. The encoded protein belongs to a group of methyltransferases capable of catalyzing the S-adenosyl-L-methionine (SAM)-dependent methylation of chloride (Cl-), bromide (Br-), and iodide (I-) to produce methyl halides.In four big causes of the plant infectivity diseases, the phytoparisite nematode's harm surpasses the bacterium and the virus, is only inferior to the fungi. Root-knot nematodes are plant-parasitic nematodes, which do great harm to the crops. The prevention method includes: The medicine (for example methyl bromide) prevented and controlled, the biological controls like bacterium and so on. I n the world, there are already many countries using the plant to prevent the nematode and obtain good results, for example: The US, Britain, India, Mexico and so on. In my experiments, I construct many kinds of vectors of three methyl halide transferases: the HOL(ATMCT) and the homologous gene THMCT and SSMCT in Thellungiella halophila and Suaeda salsa,and then do some research with them.I construct the over-expression vectors of three MCThis-pCAMBIA3301s and transform them into the Arabidopsis in order to compare the three kinds of enzyme activities in every transgenic plant.I construct vectors of three MCT-pINT4s to provide to my workmates to transform into the tobacco, soybean, strawberry and lotus corniculatus. We expect to reduce the Cl- content of the plants, improving the K+ content in tobacco using the cheap KCl and produce insect-resistant varieties by producing CH₃Br, which will improve the quality of transgenic tobacco.By constructing vectors of three location vectors of MCT-GFP-pCAMBIA3301 to transform Arabidopsis to study the enzyme location in cells and find that they are located in cytoplasm.I construct vectors of three MCT-p195s to transform yeast in order to get the reconstructed fungi that can prevent the nematode.By constructing the HOL-promoter-GUS vector, we know that the HOL gene is mainly expressed in young leaves, roots, veins and epidermal trichome, the inflorescence and the leaf bud, the corolla, the silique of Arabidopsis.Prokaryotic expression of the three MCTs indicates that ATMCT and THMCT are soluble proteins in the s upernatant s and SSMCT is inclusion bod y in the pellet. I purified the ATMCT and THMCT by the Ni-histag affinity agarose and washed the SSMCT to be pure enough to get the antibody by immuning the rabbit. By western blotting, we can see that the two antibodies are good enough.I just did the foundation work in studying the three enzymes and in the future the work will be focus on the comparing activities of the three enzymes.