Abiotic Stress Resistance of Escherichia coli Transformed with Arabidopsis thaliana AtTERT Gene

doi:10.13560/j.cnki.biotech.bull.1985.2021-0498

Abstract

Abstract:

Telomerase is a special reverse transcriptase in eukaryotes that maintains DNA integrity at the end of the chromosome. Here we studied the effects of the AtTERT in Arabidopsis thaliana on the growth and abiotic stress response of Escherichia coli,for laying a foundation in further studying the non-telomeres function of TERT protein. The pET32a-AtTERT prokaryotic expression vector was successfully constructed after the A. thaliana AtTERT gene was transferred into E. coli. The induced conditions were optimized,the GST-AtTERT fusion protein was purified and identified,then verified by Western blotting,and the abiotic stress resistance of the AtTERT in E. coli was detected by drop plate method. The optimal conditions for the expression of AtTERT protein were as follows：the induction temperature for competent cells Transetta（DE3）was 20℃,and the concentration of inducer（IPTG）was 0.5 mmol/L. The relative molecular weight of GST-AtTERT fusion protein was 156 kD. Western blotting verification results showed that the protein bands with the expected molecular weight appeared in the induced whole bacteria,in the supernatant and precipitate. The survival rate of E. coli transformed with AtTERT was significantly higher than that of empty control E. coli under NaCl（400 and 500 mmol/L）,mannitol（400 and 600 mmol/L）and hydrogen peroxide（0.4 mmol/L）stress. With 6 freezing-thawing cycles of liquid nitrogen,the survival rate of AtTERT was significantly lower than that of the empty control bacteria. The resistance of the E. coli transformed with AtTERT under NaCl salt stress,osmotic stress and hydrogen peroxide stress was enhanced. However,the resistane of the E. coli transformed with AtTERT under low temperature was reduced, indicating that A. thaliana AtTERT has non-telomere function of resisting abiotic stress.

Key words: Arabidopsis thaliana, telomerase reverse transcriptase, genetic transformation, fusion protein expression, abiotic stress

YANG Jia-hui, SUN Yu-ping, LU Ya-ning, LIU huan, LU Cun-fu, CHEN Yu-zhen. Abiotic Stress Resistance of Escherichia coli Transformed with Arabidopsis thaliana AtTERT Gene[J]. Biotechnology Bulletin, 2022, 38(2): 1-9.

Figures/Tables 9

Fig. 1 PCR amplification of AtTERT gene A：AtTERT gene with pET32a vector sequence. B：AtTERT gene with pGEX-4T-1 vector sequence. M：5 000 bp DNA marker

Fig.2 Double digestion identification of pET32a/ pGEX-4T-1-AtTERT A：Identification of pET32a-AtTERT recombinant plasmid after enzyme digestion. B：Identification of pGEX-4T-1-AtTERT recombinant plasmid after enzyme digestion. M：5 000 bp marker. 1,2：Recombinant plasmid after enzyme digestion. 3：Control plasmid

Fig. 3 Detection of GST-TERT protein by SDS-PAGE A：Expression of soluble protein under different IPTG concentrations at 20℃. B：Expression of soluble protein under different IPTG concentrations at 30℃. C：Expression of inclusion body protein under different IPTG concentrations at 20℃. D：Expression of inclusion body protein under different IPTG concentrations at 30℃. M：180 kD marker

Fig. 4 Western blotting identification of GST-AtTERT fusion protein A：Identification of GST-AtTERT fusion protein pattern（1,2：bacterial protein induced by empty vector control bacteria and transgenic recombinant bacteria;3,4：soluble protein induced by empty vector control bacteria and transgenic recombinant bacteria;5,6：inclusion body protein induced by empty vector control bacteria and transgenic recombinant bacteria）. B：Identification of purified GST-AtTERT fusion protein（1,2：soluble protein induced by empty vector control bacteria and transgenic recombinant bacteria;3：outflow after incubation;4：outflow after miscellaneous protein washing out;5-7：protein after elution）

Fig.5 Effect of AtTERT on the growth characteristics of E.coli **indicates that the difference is extremely significant at P<0.01;*indicates that the difference is significant at P<0.05. The same below

Fig.6 Effect of AtTERT on the survival rate of E. coli under salt stress A：Growth status of empty control bacteria（pET32a）and transgenic recombinant bacteria（pET32a-AtTERT）. B：Survival rate of E.coli when the bacterial solution diluted 104 times

Fig.7 Effect of AtTERT on the survival rate of E. coli under mannitol stress A：Growth status of empty control bacteria（pET32a）and transgenic recombinant bacteria（pET32a-AtTERT）on the LB solid medium containing mannitol. B：Survival rate of E.coli when the bacterial solution diluted 104 times

Fig.8 Effect of AtTERT on the survival rate of E. coli under cold stress A：Growth status of empty control bacteria（pET32a）and transgenic recombinant bacteria（pET32a-AtTERT）on the LB solid medium after repeated freezing and thawing in liquid nitrogen. B：Survival rate of E.coli when the bacterial solution diluted 104 times

Fig. 9 Effect of AtTERT on the survival rate of E. coli under hydrogen peroxide stress

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