Study On Maizetransformation Based On RNA Interference Of Endogenous Trehalase Gene

Studies have shown that many organisms can be long-term survival under harsh environmental conditions of drought, high temperature, high salinity, high concentrations of trehalose content in cells is closely related to. If the organism is more severe environmental stress, trehalose on organism and biological activity play a good role in the protection, exogenous trehalose on organism and biological macromolecules activity also plays a role in protection.In the past, people think that trehalose as a carbon source is stored in cells in vivo.In the later, found that trehalose is usually in environmental stress conditions, its content with the external environment changes, is a stress metabolite.Trehalose synthase gene in plant stress resistance in the research process is found in glutamate, proline, glycine betaine synthetase genes after a resistance related gene. Accumulation of trehalose in plants, with the resistance and protective effect of energy reserves and other biological functions, which can be improved by plant some undesirable traits, may eventually reach the optimization of breeding effect.At present, mainly through the following two methods for trehalose synthesis and metabolism pathway of interference, thereby realizing trehalose in living organisms:one is based on the accumulation of trehalose synthase gene cloning research, construct its gene expression vector, and the vector into the organism, to achieve the trehalose accumulation. Current research is also the largest such method. The other is through inhibition of trehalase, namely the trehalose hydrolase biological activity, blocking the trehalose decomposition approach, this method can by chemical means,also through biological means to achieve.Trehalase is only to hydrolysis of trehalose into glucose enzyme. In1969, trehalase found in the sugar cane seedlings, followed by a number of other plants also detected the presence of trehalase. Muller etc. study found that validamycin A could inhibit the activity of the Arabidopsis trehalase in vivo, trehalose accumulation occurs in the flowers, leaves and stems, sucrose and starch content are lower. It also confirmed that trehalose is indeed a plant endogenous substances. So to the organisms in vivo accumulation of high levels of trehalose trehalose hydrolase, we can respect proceed with, try to inhibition of trehalose degradation, but want to suppress a protease activity, RNA interference gene silencing is the effective means to. Therefore, we use RNA interference maize endogenous trehalase to corn in vivo accumulation of high levels of trehalose to.The physical and chemical properties of trehalase in particular biological characteristics of the latest research and applications are introduced in detail to provide a good reference for further study of the trehalase. Study the regulation of expression of this enzyme in corn, not only helps to make clear the physiological functions, is also expected to improve the drought tolerance of maize through genetic modification, even with high expression of the TPS gene of corn for bio-reactor to extract the production of trehalose.In this study, RT-PCR technique from corn18-599white total RNA was amplified320bp fragment of the trehalase gene W and J, and to them the pros and cons on both sides of the intron inserted into the vector pSK, the formation of a W (RNAi) components and a J (RNAi), components, by BamHI, and SmaI digestion and cloned into the plant expression vector pTF101.1the formation pTU1and pTU2carrier. Confirmed by restriction enzyme digestion and sequencing, and successfully constructed the trehalase gene inverted repeats of RNAi expression vector. By freeze-thaw method to pTUl, and pTU2carrier transferred to Agrobacterium tumefaciens EHA105. With pTU1and pTU2vector Agrobacterium tumefaciens EHA105, infect maize18-599red18-599white embryogenic callus induction after herbicide screening of transgenic plants. PCR detection and amplification of target fragments were detected, three positive transgenic regeneration strains. To plants from100seeds in Yunnan on behalf of, by PCR and Southern blotting confirmed the positive ten T1generation of transgenic renewable lines. At the same time on T1positive transgenic plants were randomly selected to do a quantitative RT-PCR detection, the results demonstrated that RNA interference in silent maize endogenous trehalose hydrolase gene and played a certain effect.