Studies On Cloning Of The Thermostable ?-amylase From The Bacillus Licheniformis And Expressing In Arabidopsis

Liquidation is an impotant technique of starch processing in factory. The common way for liquidation is mixing the exogenous thermostalbe a-amylase until the starch dissolves which not only need special equipment and technique, but also complicated. All of these result in the increassed cost of liquidation.Transforming the gene of thermostable a-amylase to plant which synthesize starch can obtain the starch materials include thermostable a-amylase. Using these raw material can save the step of adding thermostable ?-amylase, which can simplify the process and reduce the cost. For discussing the possibility of expression of the thermostable a-amylase in plant, this research cloned the full-length of the thermostable a-amylase gene from the Bacillus licheniforms strain and transformed in Arabidopsis thaliana and potato for expression so that realize the synchronized put of thermostable a-amylase and starch material.Specific findings are as follows:1. Full-length of the thermostable a-amylase gene from a Bacillus licheniforms strain was cloned in a prokaryotic expression vector and a eukaryotic expression vector and transformed the vector in E.coli and Pichia pastor is, in addition, coated plates in the LB and YPDA medium. Observed the transparent zones of medium which contained starch after dying with I2-KI solution obviously. The thermostable a-amylase was overexpressed in E.coli by IPTG induction for6hours at28?, protein bands at55KDa were detected with SDS-PAGE. Both results show that the gene of thermostable a-amylase can be expression in prokaryotic microorganism and eukaryotic microorganism, which have correct activity.2. Construsted the recombinant plasmind of plant expression vector with the gene of thermostable a-amylase and transformed into LBA4404. Green fluorescence was observed both in cytoplasm and vacuole of lower epidermis of tobacco by fluorescence microscope.Using I2-KI solution for dyeing the tobacco leaf which decolorized by alcohol can observe the different of color because the starch of the leaf of wild type can't be hydrolyse by thermostable a-amylase so that the leaf dyed to blue. The area of leaf which infected by LBA4404/G1300-a is no change, on the constrary. The result of dyeing and subcellular localization prove the gene of thermostable a-amylase can be recognized by plant and translate to a protein have correct activity.3. Using the method of floral dip introduce the gene into Arabidopsis, the homozygote of Arabidopsis which can inherit the a-amylase gene stably were screened.Detected the activity of thermostable a-amylase which expressed in the leaf of Arabidopsis with two ways. The first method is using I2-KI solution for dyeing the leaf of Arabidopsis from physicochemical point of view. The transgenic Arabidopsis has no change after dyeing with I2-KI solution because the strach has been hydrolysed by the thermostable a-amylase which contrast to the wild type Arabidopsis has been dyed into blue. The second method is analysis the change of glucose and maltose of hydrolysate by HPLC. Compared to the standards and slurry of Arabidopsis, there is a obvious peak of maltose in the spectrum of sample. The result display the thermostable a-amylase can hydrolyse the starch from metabolite. Both of the two methods confirm the expressive thermostable a-amylase of Arabidopsis has correct activity which can hydrolyse the starch directly.In order to analysis whether the thermostable a-amylase will influence the growth of plant, phenotypic analysis is necessary.Using MS medium culture both transgenic and wild type Arabidopsis. The result shows that there is no different between them which prove the expression of thermostable ?-amylase won't influence the growth of plant.4. Introducing the gene of thermostable a-amylase into potato for expressing. Comparing the CK and transgenic potato, no significant difference found between them, the obtained of transgenic potato plants lays a foundation for the further researchments that enable the expressions of thermostable a-amylase in high-starchy plants.