Study On The Interaction Proteins Of GhPDP In The Energy Metabolism Pathway Of Cotton Cytoplasmic Male Sterile Line

Cytoplasmic male sterility is an important biological trait of higher plants.The mechanism of its production has not yet been fully resolved because it is extremely complicated.Previous studies had shown that cytoplasmic male sterility was caused by the affect of the abnormal energy metabolism on the normal development of pollen.Pyruvate dehydrogenase system was a key enzyme in energy metabolism,and pollen development was related to its activity and regulation.Pyruvate dehydrogenase phosphatase PDP was the main regulatory enzyme of energy metabolism in the mitochondria of higher organisms.The reason of abnormal energy metabolism pathways was that the steady state of pyruvate dehydrogenase PDC activity regulation is broken because of the change of the regulatory enzyme genes.The catalytic activity of PDC was restored by the phosphohydrolase properties of PDP to dephosphorylate PDC.The pyruvate dehydrogenase phosphatase GhPDC-E1α,GhPDK and GhPDP genes have been cloned from cotton Jin A cytoplasmic male sterile line and its maintainer line,the bioinformatics analysis has been performed on them in our laboratory before.We also studied gene expression at the transcriptional level,and speculated that GhPDP may be related to the process of pollen abortion.In this study,the enzyme activities of GhPDC-E1α,GhPDK and GhPDP during the microspores development of the cotton Jin A cytoplasmic male sterile line and its maintainer line were measured.Combined with the results of previous studies,the effect of GhPDP on GhPDC-E1α activity was preliminarily analyzed;Prokaryotic expression system of GhPDP genes was constructed,expressed and purificated;The proteins that interact with GhPDP were detected from the total protein of the flower buds on cotton Jin A cytoplasmic male sterile line,the potential interaction protein genes was cloned,and the further interaction relationship was verified too.The results are as follows:1.The enzyme activities of GhPDC-E1α,GhPDK and GhPDP were measured during the development of the microspores of the Jin A sterile line and its maintainer line.The results showed that the enzyme activity of GhPDC-E1α from Jin A sterile line was lower than that of the maintainer line at the stage before microspore abortion;the enzyme activity of GhPDK from Jin A sterile line during the microspore abortion period was significantly lower than that of the maintainer line;the activity of GhPDP enzyme from Jin A sterile line was significantly lower than the maintainer line both the pre-abortion period and the abortion period.Studies have shown that the level of GhPDC-E1α activity in Jin A sterile line is not consisten with the amount of PDC-E1α protein expression,it depended on the activity level of its activity regulating enzyme GhPDP.2.Using the Over-Lap PCR method,the recombinant expression vector p ET-22b-GhPDP was constructed,and the induction temperature,induction time,IPTG final concentration and OD600 were optimized through single factor experiment and orthogonal experiment.The p ET-22b-GhPDP was expressed d in E.coli Transetta(DE3)and purified,GhPDP protein with a molecular weight of 42 k D was obtained.3.Using His pull-down technology,potential proteins that may have interactions with GhPDP were found and analyzed by mass spectrometry from the flower buds of the cotton Jin A cytoplasmic male sterile line.A total of 8 proteins that may interact with GhPDP protein were found according to the criteria(the peptide coverage,score,and number of peptide matches and so on).They are V-type proton ATPase catalytic subunit A,chaperone protein CPN60-2,T-complex protein 1 subunit α,acetaldehyde dehydrogenase family 2 member B4,V-type proton ATPase subunit B2,elongation factor 1α,Aspartic protease,type V proton ATPase subunit C.These candidate proteins were related to protein synthesis and degradation,energy metabolism and pollen growth and development.4.Using cotton Jin A cytoplasmic male sterile line as material,the potential interaction protein gene Gh VHA-B2 was cloned.The ORF sequence of Gh VHA-B2 gene was 1458 bp in length,It encoded 486 amino acids.Using the Over-Lap PCR method,the recombinant expression vector p GEX-4T-1-Gh VHA-B2 was constructed,expressed and purified in E.coli Transetta(DE3),and the Gh VHA-B2 protein with a molecular weight of 54 k D was obtained.5.Using the Over-Lap PCR method,the recombinant expression vectors p GEX-4T-1-GhPDC-E1αand p GEX-4T-1-GhPDK were constructed,expressed and purified in E.coli Transetta(DE3),GhPDC-E1α and GhPDK proteins were obtained,their molecular weights are 43 k D and 41 k D,respectively.6.The interaction between GhPDC-E1α,GhPDK,Gh VHA-B2 protein and GhPDP protein in vitro was verified Using GST pull-down technology.The interact between GhPDC-E1α,GhPDK,Gh VHA-B2 proteins and GhPDP protein was determined preliminarily.The results of this study indicated that GhPDP not only participated in the regulation of GhPDC,but also interacted with V-ATPase subunits,they had co-regulation effect on pollen development.7.The overexpression vector p RI101-AN-GhPDP was constructed by the method of double enzyme digestion,and the overexpression vector was introduced into Arabidopsis thaliana using the inflorescence infection method,the T3 generation transgenic Arabidopsis line was obtained.