Cloning And Expression Of GPD And GPP Genes Of Candida Glycerolgenesis In Saccharomyces Cerevisiae And Nicotiana Tabacum

Plant growth and development were strongly influenced by some abiotic stresses, suchas drought, high salt, low temperature et al., which picked up foodstuff scarcity due to theirnegative effects of decrease in plant productivity and reduction in planting area. Therefore, inorder to improve the tolerance to the mentioned abiotic stresses, researchers of the worldhave paid much attention to the research for a long time. And the development of molecularbiology and biological technology offer new methods for improving abiotic tolerance ofplant.The glycerol was an excellent osmoprotectants. However, many plants couldn'taccumulate enough glycerol in response to osmotic stress.Candida glycerolgenesis WL2002-5, which was isolated by our own lab, was able totolerate high abiotic stress and produce superfluous glycerol. Two genes of GPD and GPPencoding glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate phosphatase, twokey enzymes in glycerol synthesis, were cloned from Candida glycerolgenesis WL2002-5using PCR(polymerase chain reaction). After sequencing cloned GPD and GPP genes, it wasfound that the identity of the two genes with the corresponding genes in Saccharomycescerevisiae is up to 99%.Some primary researches were carried out focused on the two glycerol synthesis genesfrom C. glycerolgenesis WL2002-5. Firstly, the two genes were heterogeneously expressedin S. cerevisiae and then the results were detected. Secondly, recombinant Nicotiana tabacumwas constructed after introduction of the two genes, which made an important stride to gaindesirable plants tolerant to abiotic stresses.Expression vectors pYX212-GPD and pYX212-GPP containing GPD and GPPrespectively, which could expressed in Saccharomyces cerevisiae W303-1A, wereconstructed successfully. The GPD gene increased glycerol content from 6 gL^-1 to about 18gL^-1after 72 h fermentation, while GPP gene had little effects. The results showed thatintroduction of GPD gene could promote glycerol production significantly. Furthermore,Expression vectors pBI121-GPD , pCAMBIA3300-GPD and pBI121-GPP,pCAMBIA3300-GPP, containing GPD and GPP respectively, andpCAMBIA3300-GPD-GPP containing GPD and GPP genes, were constructed. And then therecombinant vectors were introduced into Nicotiana tabacum using Agrobacterium-mediatedgene transfer system. A method, which could identify transgenic food quickly, convenientlyand effectively, was established. Analysis of T0 plant demonstrated that GPD and(or) GPPgene had integrated into the genome of transformed plants using the above mentionedmethod.