Cloning And Prokaryotic Expression Of Starch Phosphorylase Gene DsSP From Dunaliella Salina

Soil salinization was a serious problem affecting agricultural production and ecologicalenvironment,and the reduction of arable land and freshwater resources forced human to developlarge areas of saline,coastal and beach areas. Therefore, the mechanism of cultivating plants andsalt tolerant plants had become a hot research.Dunaliella salina referred salina belonging to Dunaliella, Dunaliellaceae, Chlamydomonadales,Chlorophyceae,Chlorophyta. Dunaliella salina, a kind of aneukaryotic unicellular greenalgae without cell walls, could survive in the extreme environment from0.05mol/L to thesaturated concentration of salt solution,was one of the most salt-tolerant eukaryotes everdiscovered, and was also the model organisms to study the mechanism of plant salt tolerance.Over the years, many scholar study results confirmed that the rapid volume change and glycerolmetabolism played an important role in fast response to the change of salina outside osmoticpressure. But it was still not clear for the further regulation of gene expression and Signaltransduction pathway. When exposed to high salt stress, to respond to the change in the externalosmolarity, Dunaliella salina synthesized a large number of glycerol by Starch degradation.Under the action of starch phosphorylase,Starch was converted to glucose which generateglycerol after a series of ways, and the starch phosphorylase played a key role in this process.But the key genes of starch conversion had not been cloned from salina and no people revealedits basic properties.Therefore, the study of starch phosphatase has important scientificsignificance in elucidating the highly complex molecular mechanisms of salt tolerance of salina.In this study, a kind of starch phosphatase gene was cloned from Dunaliella salina and itwas analysed by Systematic bioinformatic and prokaryotic expression. The main experimentmethods and conclusions of the thesis were as fellows:1. A kind of starch phosphorylase gene (GenBank accession No.KF061044) named DsSPwas isolated from Dunaliella salina using RT-PCR and rapid amplification of cDNA ends(RACE) method,and the full-length cDNA of DsSP was3560bp, containing a48bp5’-untranslated region (UTR),a416bp3’UTR and a3096bp open reading frame (ORF) whichencoded1031amino acids.2.Bioinformatics analysis showed that: The protein was a hydrophilic andnon-transmem brane protein, which had no signal peptide and transmembrane region, and was located in thechloroplast; Secondary structure contained384α-helixs,192β-folds and455random coils;In870-882, there was a Phosphorylase pyridoxal-phosphate attachment site: EASGTSNMKFSMN;Therewere5serine phosphorylation sites,10threonine Phosphorylase horylation sites and12tyrosinephosphorylation sites; Dimensional modeling clearly showed the functional site included onlyα-helix and random coil; Phylogenetic analysis showed that it was closest with Chlamydomonasand Volvox starch Phosphorylase. 3. The prokaryotic expression vector pGS21a-DsSP was successfully constructed byconnecting the open reading frame of DsSP and the plasmid pGS-21a.Then it was transformedinto E.coliBL21, and was induced by isopropyl-β-D-thiogalacropyranoside (IPTG).The fusionprotein existed in both soluble and inclusion body. The high purity soluble fusion protein wasacquired by purified supernatant protein, and its concentration was determined by the Bradfordto be about200μg/mL. Western blot identification confirmed that the purified fusion protein canbe specifically recognized by anti-GSTantibody and this indicatesd that the fusion protein wassuccessfully expressed in E.coli.BL21.This study successfully cloned a kind of starch phosphorylase gene DsSP from Dunaliellasalina, and then analyed it by using bioinformatics, and acquired a large quantity of highlypurified protein by the prokaryotic expression method. These results laid a foundation for thestudy about the role of DsSP in salt tolerance mechanism of Dunaliella salina at the protein level next step.Simultaneously,since the gene was longer,extening from intermediate section to the5’end when cloning,hoped to provide an idea for researchers who might encounter the similarproblem.