The Thermophilic Adaptation Mechanism Of Cmr1 And Cmr4 In Thermoanaerobacter Tengcongensis

The tte2635 and tte2633 of Thermoanaerobacter tengcongensis were annotated as cmr1 and cmr4,respectively,belonging to the CRISPR-Cas III-B system.In previous studies,it was found that CRISPR-related genes were transcriptionally up-regulated in Thermoanaerobacter tengcongensis under high temperature culture conditions.The research on the correlation between the CRISPR system and the thermophilic mechanism has not been reported.This thesis conducted the following research to explore its thermophilic mechanism.1)Analysis of CRISPR structure in thermophiles.The whole genome of 61 strains of thermophiles was downloaded from Gene Bank,and the relationship between CRISPR structural characteristics of thermophiles and the degree of thermophilia was analyzed using software.The results showed that 59 of the 61 thermophiles contained CRISPR loci and the CRISPR III system accounted for the most.The relationship between the average length of CRISPR loci and the average number of spacer sequences in thermophiles was: general thermophiles>medium thermophiles>extremely thermophiles.The relationship between the minimum free energy of RNA secondary structure corresponding to repeats and the average number of CRISPR loci was: extreme thermophiles>medium thermophiles>general thermophiles.The average length of CRISPR loci and the average number of spacer sequences were negatively correlated with the degree of thermophilicity.The minimum free energy of RNA secondary structure corresponding to repeats and the average number of CRISPR loci were positively correlated with the degree of thermophilicity.there was a certain relationship between the CRISPR structural characteristics of thermophiles and the degree of thermophilicity.2)Analysis and verification of thermophilic correlation between cmr1 and cmr4 genes and Thermoanaerobacter tengcongensis.The cmr1 and cmr4 were cloned using PCR.Then the prokaryotic expression vector p ET-28a::cmr1 and p ET-28a::cmr4were constructed to express Cmr1 and Cmr4 in E.coli BL21.Bioinformatics analysis found that the two proteins are hydrophilic proteins,and neither have a transmembrane structure.The difference in expression of cmr1 and cmr4 were analyzed at 50℃,60℃,75℃ and 80℃ by fluorescence quantitative PCR.The expression levels of cmr1 and cmr4 were low at 50℃ and 60℃,and very high at 75℃and 80℃.The results showed that these two genes play an important role under high temperature conditions.Deletion plasmids of cmr1 and cmr4 genes were constructed using homologous recombination and were transformed into Thermoanaerobacter tengcongensis to construct deletion strains Δcmr1 and Δcmr4.To cultivate them and draw growth curve at 50℃,60℃,75℃ and 80℃.The growth curve was measured and it was found that Δcmr1 and Δcmr4 did not grow at 80°C,and grew slowly compared with WT at 60°C and 75°C.All three strains grow very slowly at 50℃ The results showed that these two genes play a role in the thermal adaptation process of Thermoanaerobacter tengcongensis.