The Cloning And Ectopic Prokaryotic Expression Of Suaeda Salsa CYP-1gene CDNA Involved In Salt Tolerance

Soil salinization seriously harms the agricultural development in the world andendangers the survival of human beings, which becomes the major environmental problem.In order to develop salty soil resources of and enhance the crop production with theapplication of biochemistry and molecular techniques, the salt-tolerant gene are isolatedand cloned from salt-tolerant crops and transformed into other high-yield crops with lowsalt tolerance, making them grow healthily under the high-salt pressure. Cyclophilins aredistributed extensively in the cytoplasm and miscellaneous organelles with the PPIaseactivity, which can catalyze cis-trans isomerization process and accelerate protein foldingaccurately. The results indicated that the enzyme activity was closely related to saltresistance and the over-expressed cyclophilin strikingly enhanced their tolerance capacityof the salt stress. Suaeda salsa is a typical indicator of the saliferous fields. In this paper,the gene encoding cyclophilin SsCYP-1was cloned and over-expressed in E. coli(Escherichia coli) and then the salt tolerance activity in the LB culture medium wasanalyzed. The main results in the paper were listed as follows:1) The molecular characterization of the SsCYP-1geneThe full-length SsCYP-1gene was cloned and assembled from Suaeda salsa with thedegenerate primers and RACE methods. The full-length SsCyP gene sequence was875bpin size with the open reading frame (ORF) of531bp in size. The ORF encoded176aminoacids with the predicted molecular mass of18.8KDa. The isoelectric point was7.67withthe high abundance of Gly and Lys, which owned21basic amino acids (K, R),20acidicamino acids (D, E),54hydrophobic amino acids (A, I, L, F, W, V) and39hydrophilicamino acids (N, C, Q, S, T, Y)。This sequence exhibited high homology with Ziziphusjujuba and Capsella rubella with the88%identity. The secondary structure of the SsCYP-1protein was composed of the alpha helixes (19.89%), the random coils (40.34%), theextended strands (28.41%) and a few β-turns (11.36%). The hydrophobicity analysisshowed that the protein exbihited5phosphorylation sites and shared considerablehydrophilicity at both ends but no detectable signal peptides and coil-coils.2) SsCYP-1gene expression vector construction for the heterogenous expressionAccording to the obtained SsCYP-1gene sequence, the primers with BamH I and NotI sites included were used to amplify the entire sequence. The prokaryotic expression vectors pET-dute-1/SsCYP-1was constructed and transformed to E. coli Transetta(DE3).SDS-PAGE analysis indicated that the molecular mass of these expressed protein was inaccordance with the predicted molecular weight with no corresponding bands in negativecontrol, which verified that the SsCYP-1protein expression vector was constructedcorrectly.3) The salt tolerance activity determination of the SsCYP-1proteinThe identical volumes of recombinant strains (pET-dute-1/SsCYP-1) and native strains(pET-Duet-1) were inoculated into the LB culture (0.01mM IPTG,50mg/L ampicillin) atdifferent salt concentrations (1%,6%,7%,8%NaCl) and the absorbance of both LBcultures was determined under the OD600to detect the growth state. The results showedthat the recombinants (pET-dute-1/SsCYP-1) displayed better salt-tolerance in comparisonwith the control, which suggested that the SsCYP-1genes might play an important role inplant defense against the salt stress.