Analysis Of The Activity Of The Aspartic Protease And The Interaction With ARC1 Of Ornamental Kale

Self-incompatibility(SI)is a mechanism in which a male and female fertile plant cannot produce seeds after self-pollination.Most cruciferous plants exhibit self-incompatibility,which allows stigma to recognize and distinguish "self" pollen,thereby preventing self-fertilization and inbreeding.At present,ARC1(Arm repeat containing 1)is the SI signaling factor in stigma papillary cells,and ARC1 has the activity of E3 ubiquitin ligase,which degrades its downstream substrate through the ubiquitin/26S proteasome pathway,resulting in SI reaction.In order to further analyze the ARC 1-mediated SI reaction mechanism,we obtained a protein interacting with ARC1 using the yeast CytoTrap two-hybrid library.In the studies,the full-length cDNA of BoASP1 was obtained by RACE method,and BoASP1 fusion protein was obtained by prokaryotic expression purification technology.The fusion BoASP1 protein was analyzed for aspartic protease enzyme activity.The expression of ASP1 in the various tissue and different developmental stigma were detected by immunoblotting.Finally,the interaction domain of ASP1 and ARC1 were analyzed by yeast two-hybrid.The main results are as follows:1.BoASP1 cDNA was 1596 bp in the full length,which contained a 5'-untranslated region(5'-UTR)of 69 bp,a 3'-untranslated region(3'-UTR)of 144 bp and an opening reading frame of 1383 bp encoding a 460 predicted amino acids residue protein.The deduced amino acid sequence of BoASP1 shared 97%and 93%identity with Brassica napus BnASP1 and Brassica rapa BrASP1,respectively.2.The BoASP1 coding region sequence was amplified and inserted into the prokaiyotic expression vector pET-14b to obtain the pET-14b-BoASP1 expression plasmid.The E.coli BL21(DE3)cell was transformed pET-14b-BoASP1.SDS-PAGE results showed that the recombinant BoASP1 protein about 58 kDa was induced by IPTG and purification by affinity chromatography using Ni2+-NTA resin.The polyclonal antibodies against BoASP1 were prepared.3.The expression of BoASPl in the various tissue was examined.The total protein of sepals,petals,stamens,stigmas,styles,and ovary were extracted.The results of immunoblotting showed that BoASP1 was expressed in all tissues and was highly expressed in stigma and style.To further analyze the expression level of BoASP1 in different developmental stigma,the total protein in the stigma S1-S5 period were extracted.The results of immunoblotting showed that BoASP1 did not change significantly during the stigma development.4.BoASP1 was constructed to the prokaryotic expression vector pCold,and the pCold-BoASPl plasmid was obtained,the soluble protein BoASP1 was purified.The relative enzyme activity of the protein was determined by FITC-labeled casein method.The results showed that the activity BoASPl gradually increased in 0-300 ng range,and the highest proteolytic activity to 300 ng reached the highest.The proteolytic activity of 300-500 ng gradually decreased.5.Two active sites of BoASP1,DTG and DSG,were mutated to asparagine,and a mutant vector pCold-BoASP1-M2 was constructed.After transformation and induction,the soluble protein BoASP1-M2 was obtained by affinity purification.Enzyme activity analysis showed that ASP1-M2 had no proteolytic activity in the range of 0-500 ng.The results showed that the activity of BoASPl protein depended on the active sites of DTG and DSG.6.To analyze the interaction of the BoASP1 domain with BoARC1,pMyr-BoASP11-460,pMyr-BoASP 166-460,pMyr-BoASP 1 190-460,pMyr-BoASP 1 280-460,pMyr-BoASP1 390-460 and pMyr-BoASP11-460-M2 were constructed.The results of CytoTrap yeast two-hybrid analyses showed that BoASP1390-460 interacts with BoARCI,while BoASP166-460,BoASP1 190-460,BoASP1280-460,BoASP1 1-460,BoASP1-M2 do not interact with BoARC1.BoASP1390-460 was identified as a key domain that interacts with BoARC1.7.To analyze the interaction between BoARCl and BnARCl domains and BoASP1390-460,pSos-BoARC1 1-279,pSos-BoARC1 1-361,pSos-BoARC1280-361,pSos-BoARC 1280-663,pSos-BoARC 1362-663,pSos-BnARC1-280,pSos-BnARC1 1-359,pSos-BnARC1281-359,pSos-BnARC1281-661,pSos-BnARCI36CV661 and pSos-BfnARC11-661.The results of CytoTrap yeast two-hybrid analyses showed that BoARC1280-663 and pSos-BnARC1281-661,U-box domain and ARM domain of ARC1,interacted with BoASP1390-460,while BoARCl 1-279,BoARC11-361,BoARC1280-361,BoARCl362-663 does not interact with BoASP1390-460.The results show that the C-terminus of ARC 1 mediates the interaction with ASP 1.