Cloning and Prokaryotic Expression of the GhMYB42 Gene in Cotton
MYB transcription factors play an important role in the growth and development of cotton,and GhMYB42 is one of the transcription factors of the MYB family,which also has certain research value,so this study cloned the cod-ing sequence of GhMYB42 gene from cotton and constructed a prokaryotic expression vector.The nucleotide sequence and amino acid sequence of GhMYB42 were analysed using bioinformatics methods,and the coding sequence of the Gh-MYB42 gene was constructed into the prokaryotic expression vector pGEX-4T-1 by the Gataway BP and LR reactions,and the optimal conditions for the induction of the protein by IPTG were determined by setting up different IPTG-inducing conditions,and finally the recombinant protein was identified by Western Blot.The results showed that the full-length se-quence of GhMYB42(XP_016732693.1)was 1 508 bp,the coding region length was 792 bp,the coding area was 263 ami-no acids,the predicted molecular weight was about 29.534 ku,and the isoelectric point was 5.18.Amino acid sequence comparison analysis revealed that the sequence similarity of the MYB transcription factors was 80.62％,and that the N-terminus of the GhMYB42 protein contained two tandem SANT structural domains,making it an R2R3 transcription fac-tor.Phylogenetic tree analysis showed that the MYB42 protein in Gossypium hirsutum had the highest homology with an-other MYB protein(XP_012439547.1)in Gossypium hirsutum and was on one branch.Since the results of each ex-perimental gradient were not significantly different during protein induction,it chose the conditions for IPTG to be 0.2 mmol/L at a final concentration of 0.2 mmol/L,a temperature of 37 ℃ for 3 h,and a temperature and time for pro-tein solubility to be induced at 37 ℃ for 3 h.The Western Blot results showed that the size of the recombinant protein was correct,and finally the GhMYB42 recombinant protein with a size of 55.54 ku was successfully obtained,and it will purify the recombinant protein and further study the function of the transcription factor GhMYB42.
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维普
