Effects Of MCT Gene Heterologous Expression On Stress Tolerance Of Plants

As a kind of highly effective soil fumigant, methyl bromide is extensively used in facility agriculture around the whole world. But it plays the destructive effect to the ozone layer and the environment. Therefore, it is necessary to research on source and sink of methyl halides, the transport agent of halogen to the atmosphere. The development of alternative technologies for methyl bromide is imminent, before it is eliminated by the United Nations Montreal Convention in2015.Despite the economic and environmental important value of methyl halides, the natural sources and biological production mechanisms studied little. Besides CH3Br fumigation, important sources include oceans, biomass burning, tropical plants, salt marshes and certain crops and fungi. The methyl halide is biosynthesized by an enzymatic reaction:SAM+X-(Cl-, Br-, or I-)â†'CH3X+S-adenosylhomocysteine. This reaction is catalyzed by one kind of methyl transferase (methyl chloride transferase MCT), a biological catalyst responsible for the production of atmospheric methyl chloride, is a novel enzyme found in several fungi, marine algae, and halophytic plants. A survey based on CH3I production by leaf discs supplied with KI detected that the halide methyltransferase is ubiquitous actively in higher plants. Whether or not the halide methyltransferase is related to naturally occurring salt tolerance, it may be confers 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.The phytoparisite nematode is a widespreaded harmful pathogens causing the plant infectivity diseases,which surpasses the bacterium and the virus, is only inferior to the fungi.In certain areas, the prevailing nematodes are highly destructive, promoting fungi and bacteria infect plants easily and eventually leading to plant death. The prevention method includes:the medicine (for example methyl bromide) prevented and controlled, the biological controls like bacterium and so on.According to the nature that higher plants can produce methyl halides, the gene of methyl chloride transferase——AtMCT,ThMCT and SsMCT, were cloned, and was transformed into tobacco. The results of Southern and Northern blotting showed that the genes expressed heterologously in tobacco.After treated with different concentration solutions, indexes such as release amount of halogenated methane, contents of Cl-, Na+, K+and chlorophylls, and biomasses of wild type and transgenic plants were determined. And the results showed that the release amount of CH3I, CH3Cl or CH3Br from transgenic tobacco plants was markedly higher than from wild-type tobacco. When treated with different concentrations of Na+or Cl-, the transgenic tobacco plants had more tolerance to Na+or Cl-, containing higher contents of chlorophyll, lower Na+and higher K+.We focus on improving the K+content and reducing the Cl-content in tobacco by using the economical and practical KCl fertilizer and produce insect-resistant varieties by producing CH3Br, which will improve the quality of transgenic tobacco.Prokaryotic expression of the three MCTs indicates that AtMCT is soluble in the supernatant, SsMCT is a inclusion body in the pellet and half of ThMCT is hydrophilic while the other half is hydrophobic. In order to search for the different solubilities of the MCTs, ThMCT were reconstructed by point mutations, and the recombinant proteins were all changed soluble in the supernatant.AtMCT-His, ThMCT-His, ThMCTA32E-His, ThMCTL49Q-His, ThMCTA32EL49Q-His and SsMCT-His were purified by the Ni-histag affinity agarose and washed to be pure enough to get a single stripe in SDS-PAGE.The purified methyl chloride transferase activity was measured by the methyl halides production released from the reaction. Results revealed that the SsMCT activity was high when the reaction was carried out with only enzyme and KI. In4M NaCl, the synthetic activity of iodine of the three enzymes was inhibitd, because chloride is a competitive inhibitor of iodide. AtMCT and ThMCT synthesis methyl chloride specifically. The three recombinant enzymes activity was influenced by salt concentration. LQMCT and AEMCT activities for iodide were higher. Meanwhile, AELQ can colerate higher concentration of salt. The result illustrates that the two amino acid sites are important in regulating the enzyme activity.The overpression of AtMCT in tobacco confers more resistence of transgenic plants to Na+, Cl-and nematode, which indicates that cultivation of AtMCT transgenic crops is expected to replace CH3Br fumigation and will be a novel way for biological control of insect pests.