拟南芥AtTERT对大肠杆菌非生物胁迫抗性的影响

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

摘要/Abstract

摘要：

端粒酶是真核生物中维持染色体末端DNA完整性的一类特殊逆转录酶,研究拟南芥AtTERT对大肠杆菌生长及非生物胁迫的影响,为深入研究TERT蛋白非端粒功能奠定基础。将拟南芥AtTERT转入大肠杆菌,成功构建pET32a-AtTERT原核表达载体,优化诱导条件,纯化并鉴定GST-AtTERT融合蛋白,运用Western blotting验证,同时采用点板法检测转AtTERT大肠杆菌的非生物胁迫抗性。结果表明,优化诱导条件为感受态细胞Transetta（DE3）诱导温度20℃、诱导剂（IPTG）浓度为0.5 mmol/L; 纯化的GST-AtTERT融合蛋白相对分子量大小为156 kD;Western blotting验证结果显示在诱导全菌、上清及沉淀中均出现并与预期分子量大小结果一致的蛋白条带。转AtTERT重组菌在NaCl（400和500 mmol/L）、甘露醇（400和600 mmol/L）和H₂O₂（0.4 mmol/L）的LB固体培养基上的存活率显著高于空载对照菌,而利用液氮反复冻融6次时,转AtTERT重组菌存活率显著低于空载对照菌。转AtTERT大肠杆菌NaCl盐胁迫、甘露醇渗透胁迫、过氧化氢氧化胁迫抗性增强,但低温抗性减弱,表明拟南芥AtTERT具有抗非生物胁迫的非端粒功能。

关键词: 拟南芥, 端粒酶逆转录酶, 遗传转化, 融合蛋白表达, 非生物胁迫

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

杨佳慧, 孙玉萍, 陆雅宁, 刘欢, 卢存福, 陈玉珍. 拟南芥AtTERT对大肠杆菌非生物胁迫抗性的影响[J]. 生物技术通报, 2022, 38(2): 1-9.

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.

图/表 9

图1 AtTERT的PCR扩增 A：带有pET-32a载体序列的AtTERT;B：带有pGEX-4T-1载体序列的AtTERT。M：5 000 bp DNA marker

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

图2 pET32a/ pGEX-4T-1-AtTERT重组质粒的酶切鉴定 A：pET32a-AtTERT重组质粒酶切鉴定;B：pGEX-4T-1-AtTERT重组质粒酶切鉴定。M：5 000 bp marker;1、2：重组质粒酶切;3：对照质粒

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

图3 GST-TERT蛋白的SDS-PAGE检测 A：20℃不同IPTG浓度下可溶性蛋白的表达;B：30℃不同IPTG浓度下可溶性蛋白的表达;C：20℃不同IPTG浓度下包涵体蛋白的表达;D：30℃不同IPTG浓度下包涵体蛋白的表达。M：180 kD marker

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

图4 Western blotting鉴定GST-AtTERT融合蛋白 A：鉴定GST-AtTERT融合蛋白的表达形式（1,2：空载体对照菌及转基因重组菌诱导后全菌蛋白;3,4：空载体对照菌及转基因重组菌诱导后可溶性蛋白;5,6：空载体对照菌及转基因重组菌包涵体蛋白）;B：鉴定纯化后GST-AtTERT融合蛋白（1,2：空载体对照菌及转基因重组菌诱导后可溶性蛋白;3孵育后流出液;4：杂蛋白洗脱后流出液;5-7：纯化后蛋白）

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）

图5 AtTERT对大肠杆菌生长特性的影响 **：表示在P<0.01水平差异达到极显著水平;*：表示在P<0.05水平差异显著。下同

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

图6 盐胁迫下AtTERT对大肠杆菌存活率的影响 A：空载对照菌（CK）和转基因重组菌（pET32a-AtTERT）在LB（0 mmol/L NaCl）及含有NaCl（400和500 mmol/L）的LB固体培养基上生长状况;B：大肠杆菌菌液稀释104倍时存活率

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

图7 甘露醇胁迫下AtTERT对大肠杆菌存活率的影响 A：空载对照菌（pET32a）和转基因重组菌（pET32a-AtTERT）在含有甘露醇的LB固体培养基上生长状况;B：大肠杆菌菌液稀释104倍时存活率

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

图8 低温胁迫下AtTERT对大肠杆菌存活率的影响 A：空载对照菌（pET32a）和转基因重组菌（pET32a-AtTERT）经过液氮反复冻融后在LB固体培养基上生长状况;B：大肠杆菌菌液稀释104倍时存活率

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

图9 H2O2胁迫下AtTERT对大肠杆菌存活率的影响 A：空载对照菌（pET32a）和转基因重组菌（pET32a-AtTERT）在含有0 mmol/L过氧化氢LB固体培养基上生长状况;B：大肠杆菌菌液稀释104倍时存活率

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

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