Cloning The SacB Gene Of Bacillus Subtilis And Its Transformation

Levansucrase, coded by Bacillus subtilis SacB gene, can catalyze the hydrolysis of sucrose and fructose polymerizing into fructan. Fructan is localized in vacuole of plant cell, accumulation and degradation of fructan release large amount of free fructose to reduce the freezing point of water. As osmosis substance and cryoprotective agent of vacuole, fructan enhance the drought, salt, cold resistance of plant.In this research specific primers were designed according to encoding sequence of SacB gene from Bacillus subtilis that is searched from GenBank. A fragment containing 1500bp coding region of SacB gene was obtained and cloned by PCR. Signal peptide of patatin coding sequence was also cloned from potato in order to chimera it with the SacB and make the SacB gene expression in vacuole in the transformed cells. The SacB gene and signal peptide coding sequence of patatin are recombinated into Ti plasmid thus a recombination vector pWM101-pat-sacB was constructed. The chimera gene is located in downstream of the promoter CaMV 35S.The recombinator was first transformed into tobacco(Nicotiana tabacum) cultivar WS38 via leaf disc infected with Agrobacterium tumefaciens. Several transformed plantlets were identified and the function of SacB gene and patatin signal peptide verified. It showed that the SacB and the patatin are functional in the transformed tobacco. And then the recombination is transformed into pepper cultivar H8214 with the same method. Transfomed 3 seedlings are obtained through antibiotics selection and molecular detection. Its anbiotic stress tolerance is under testing.