Polymorphism of Rv3303c-Rv3304 of Mycobacterium tuberculosis modern Beijing genotype strains and physiological significance in China
Objective To analyze the polymorphism of gene spacer region of Rv3303c-Rv3304 of 141 strains of Mycobacterium tuberculosis in China and discuss the physiological significance of Rv3303c-Rv3304 gene spacer.Methods The sequence of Rv3303c-Rv3304 gene spacer in 141 strains of M.tuberculosis modern Beijing genotype was analyzed by polymerase chain reaction(PCR)and sequencing,and the recombinant plasmid of Rv3303c-Rv3304 gene spacer was constructed,and the recombinant plasmid and empty plasmid were electroporated into Mycobacterium smegmatis mc2155.β-galactosidase(lacZ gene expression product)activity assay was used to detect the expression level of reporter gene lacZ.Software SPSS 25.0 was used to conduct a one-way analysis of variance.Results There were 376 bp,260 bp and 144 bp sequences in the spacer region of the Rv3303c-Rv3304 gene of 141 strains of the modern Beijing genotype,which contained 4,2 and 0 copy repeats,respectively.Two copy repeats were the predominant sequence,accounting for 95.74％.Based on these three different copy repeats,recombinant plasmids pSD5B-334,pSD5B-332,and pSD5B-330 were constructed,and the recombinant plasmids and empty plasmids were transferred into Mycobacterium smegmatis.The results ofβ-galactosidase activity detection showed that the β-galactosidase activity of recombinant strains MSMEG-334,MSMEG-332 and MSMEG-330 increased compared with strains MSMEG-pSD5B,and the differences of β-galactosidase activity among strains were significant(P＜0.001).Conclusion In China,the gene spacer region of Rv3303c-Rv3304 of the modern Beijing genotype strain of M.tuberculosis,has polymorphism,which can significantly affect the transcription of reporter gene lacZ indicated by model strain verification.
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