Study On The Expression And Interaction Protein Of GhOTUD5 Gene In Cotton

Male sterility is the basis of the plant hereosis utilization,therefore it is important for plant genetics and breeding to research its mechanism.Being an important biological trait of higher plants,cytoplasmic male sterility is extremely complex,and has not been completely resolved.It is firmly believed that the expression of fertility-related genes in a certain time and space and the complex physiological and biochemical processes were involved in its formation.Being the central link in regulating gene expression aspect,covalent modification of histones,especially the level of ubiquitination and deubiquitination,is closely related to the reproductive development of plants,with the relationship to fertility studied in Arabidopsis,wheat,rice and other plants,but less in cotton.We have already obtained histone deubiquitinase GhOTUD5 gene,and performed the bioinformatics analysis and expression at transcription level by using cotton Jin A cytoplasmic male sterile line and its maintainer as the materials.In this study,we constructed a prokaryotic expression system of GhOTUD5 gene,performed the expression and purification of it,selected the interacted proteins from the total protein of cotton Jin A and verficated their interaction relations.The results were as follows:1.The recombinant expression vector pET-22b-GhOTUD5 was constructed by the way of POE-PCR and expressed in E.coli Transetta(DE3).The GhOTUD5 protein was purified and identified by mass spectrometry.The molecular weight was 59.481 kD.2.Using His pull-down technology,the protein interacting with GhOTUD5 was selected from the total protein of Jin A and analyzed by mass spectrometry.A total of 8 proteins that may interact with the GhOTUD5 protein were found according the peptide coverage,scores,and number of peptide matches in mass identification results.They were the V-type proton ATPase catalytic subunits A,T-complex protein 1subunit alpha,T-complex protein 1 subunit zeta 1,T-complex protein 1 subunit eta,T-complex protein 1subunit theta,V-type proton ATPase subunit B1,V-type proton ATPase subunit B2 and V-type proton ATPase subunit D,which could be divided into three categories: V-type proton ATPase catalytic subunit,T-complex protein 1 subunit and V-type proton ATPase subunit.3.The potential genes of interaction proteins were obtained by homology cloning strategy from Jin A.The cDNA sequence length of GhVHA-D gene was 786 bp,encoding a polypeptide containing 261 amino acid residues.A recombinant expression vector pGEX-4T-1-GhVHA-D was constructed by the way of POE-PCR and expressed in E.coli Transetta(DE3).The GhVHA-D protein was purified and identified by mass spectrometry.The molecular weight of GhVHA-D was 29.262 k D.GST pull-down technology was used to verify the interaction between GhVHA-D protein and GhOTUD5 protein in vitro.It was preliminarily determined that the GhVHA-D protein was interacted with the GhOTUD5 protein.4.Using homologous cloning,the full-length cDNA of the lysine-specific histone demethylase GhLDL1 gene was cloned and bioinformatics analysis was performed.The full-length cDNA of the GhLDL1 gene from Jin A was 2337 bp,encoding a polypeptide containing 778 amino acid residues whose molecular weight was 85.487 k D.As a hydrophilic protein with no signal peptide,GhLDL1 was transmembrane and unstable.It was predicted that GhLDL1 protein played a major role in the cytoplasm through subcellular localization,but less amount of GhLDL1 protein was detected in the nucleus and chloroplast.A recombinant expression vector p GEX-4T-1-GhLDL1 was constructed by the way of POE-PCR and expressed in E.coli Transetta(DE3).The GhLDL1 protein was purified and identified by mass spectrometry.The molecular weight of GhLDL1 protein was 85.487 k D.GST pull-down technology was used to verify the interaction between GhLDL1 protein and GhOTUD5 protein in vitro.It was preliminarily determined that the GhLDL1 protein was interacted with the GhOTUD5 protein.5.The over-expression vector pRI101-AN-GhOTUD5 was constructed by the double-digestion method.T1 and T2 transgenic Arabidopsis line were obtained through introducing the over-expression vector into Arabidopsis thaliana by Agrobacterium-mediated inflorescence infection.