Functional Identification And Heterologous Expression Of Cold Shock Proteins In Deinococcus Gobiensis Ⅰ-0

Temperature is an important environmental stress factor. A number of small cold shock proteins (Csps) are induced in response to low temperature and improve the resistance to cold stress, but little attention has been paid to the function of Csps in respons to other stresses, such as ultraviolet (UV) irradiation and osmotic stress. Deinococcus gobiensis I-0 originates from the upper layer of the Gobi desert experiencing extreme temperature differences and strong solar radiation. D. gobiensis showed remarkable resistance to UV, ionizing radiation and desiccation. In this study, to illustrate the mechanism of the Csp in increasing organisms resistance to multiple abotic stresses in Escherichia coli, two Csp genes were cloned in Deinococcus gobiensis I-0 and their function were investigated.The genes csp1 and csp2 were identified in the complete genome sequence D. gobiensis. The predicted amino acid sequences were 64% and 69% identical to the Bacillus subtilis cold shock protein CspB. Tertiary structures predicted by homology modeling using Swiss-model showed that both proteins formed a typical barrel consisting of five anti-parallelβ-strands containing an oligonucleotide binding domain for RNA or ssDNA.The D. gobiensis csp1 and csp2 were PCR amplified, resequenced, cloned into the expression vector pET28a, and introduced into Escherichia coli BL21 (DE3). Low level expression of Csp1 or Csp2 enhanced the tolerance to repeated freezing and thawing, and improved cell growth at low temperature. At higher expression levels Csp1 or Csp2 retarded cell growth. A gel mobility-shift assay showed that Csp1 and Csp2 bound ssDNA as was known for CspB, proving that the N-terminal His6-tagged proteins were functional.Csp2, but not Csp1, also increased UV resistance two-fold and osmotic resistance to 4 M NaCl or 3 M sorbitol more than two-fold. Quantitative RT-PCR assay showed increased expression after 150 J/m2 UV of genes involved in DNA base excision repair pathway including nei encoding endonuclease VIII (5-fold). This suggested that Csp2 could also improve excision repair.The transcripton of betA, betB and betT (betaine synthesis) were 1000-fold up-regulated in the Csp2-expressing E. coli after 1 M NaCl shock. The trehalose synthesis genes otsA and otsB were four-fold up-regulated, and the trehalose degradation genes treB and treC were two-fold down-regulated. This indicated that Csp2 also enhanced accumulation of osmoprotectants betaine and trehalose.To investigate the effect of Csp2 expression on stress tolerance in transgenic plants, Csp2 was expressed in Nicotiana tobacco under the control of the constitutive CaMV 35S promoter using an Agrobactium Ti vector. PCR, southern-blot and western-blot analyses revealed that the csp2 gene had been integrated into the genome and was expressed. The transgenic tobacco grew much better than the wild-type control on 20% PEG6000. This showed that the expression of csp2 improved drought tolerance in plants. These results indicated that Csp1 and Csp2 of D. gobiensis can play an important role in the respose to freezing-thawing, cold resistance and nutrition starvation of E. coli. Csp2 also increased the resistance of UV and osmotic stress in E. coli and, amazingly, the expression of csp2 also conferred drought tolerance in tabacco. It attempted to provide functional genes and substantial theoretical basis for applications of the csp gene in transgenic plants.