| Exon Junction complex(EJC)is located in the nucleus,which participates in the splicing process of introns and plays an important regulatory role in the process of plant growth and development.In this study,ornamental kale(Brassica oleracea var.acephala)was used as the experimental material,which belongs to Brassica spp.in the family Cruciferae and is an important ornamental and edible plant in northern China.The stigma receives the pollen and plays an extremely important role in plant reproduction.In order to further explore the mechanism of EJC and its core peripheral interaction proteins in the regulation of stigma development,we isolated the EJC core protein genes MAGO,elF4AⅢ,UAP56,and EJC peripheral protein genes GRP,CP29A,and obtained the recombination proteins using the prokaryotic expression technology.Moreover,we analyzed the expression of BoMago.BoeIF4AⅢ,BoUAP56 genes during stigma development by real-time quantitative PCR,and the protein expression of BoMago,BoeIF4AⅢ,BoUAP56 by immunoblotting.Finally,we finally analyzed the interactions between proteins by yeast two-hybrid experiment.The main research results obtained are as follows:1.The length of the BoMaago open reading frame is 447 bp,encoding 148 amino acids.the length of the BoeIF4AⅢ open reading frame is 1227 bp,encoding 408 amino acids.the length of the BoUAP56 open reading frame is 1284 bp,encoding 427 amino acids.The length of the BoGRPIA open reading frame is 504 bp,encoding 167 amino acids.the length of the BoGRP2A open reading frame is 510 bp,encoding 169 amino acids.the length of the BoCP29A open reading frame is 975 bp,encoding 324 amino acids.2.BoMago has 99%amino acid sequence identity with Brassica napus BnMago and Brassica rapa BrMago.BoeIF4AⅢ has 99%amino acid sequence homology with BneIF4AⅢand BreIF4AⅢ.BoUAP56 has 95%amino acid sequence homology with BnUAP56,it is also has 97%amino acid sequence homology with BrUAP56.BoGRP1A is 99%amino acid sequence homologous with BnGRP1A and BrGRP1A.BoGRP2A has 95%amino acid sequence homology with BnGRP2A and BrGRP2A.BoCP29A has 99%amino acid sequence homology with BnCP29A,it is also has 96%amino acid sequence homology with BrCP29A.3.The constructed pET-14b prokaryotic expression vector was transformed into BL21(DE3)competent cell and induced by IPTG,and then we detect the induced expression by SDS-PAGE.The protein was purified by affinity chromatography,after that we have the recombinant protein.Protein SDS-PAGE showed that the size of BoMago,BoGRP1A,and BoGRP2A proteins were around 17 kDa.The size of BoCP29A protein was around 38 kDa.The size of BoeIF4AⅢ protein was around 44 kDa,and the size of BoUAP56 protein was around 47 kDa.Purify the recombinant protein after dialysis and immunize mice to obtain polyclonal antibodies.4.The expression level of BoMago gene has the highest expression level in S1 period,which is about 3.9 times higher than that in S5 period,and the expression level in S5 period is very low.The expression level of BoeIF4A Ⅲ gene continued to decrease with the development of the stigma,and the highest expression level in S1 period was about three times that in S5 period,and it has the lowest expression level in S5 period.The expression level of BoUAP56 had the highest expression level in the S1 period.The S5 period decreased about 4 times compared with the S1 period.5.During the S1-S5 period of stigma development,we analyzed the expression levels of BoMago,BoeIF4AⅢ,and BoUAP56 proteins,it was found that these three proteins all had the highest expression at S1 period.These proteins all had the lowest expression in the S5 period.This suggests that they are acting at the early stage of stigma development.6.The vectors of AD-BoMago,AD-BoGRP1A,AD-BoGRP2A,AD-BoCP29A,BD-BoeIF4AⅢ and BD-BoUAP56 was constructed.BoMago interact with BoeIF4AⅢ,and BoeIF4AⅢ interact with BoGRP1A.BoeIF4AⅢ can not interact with BoUAP56 and BoCP29A.BoUAP56 interact with BoGRP1A and BoCP29A.BoUAP56 can not interact with BoMago,BoeIF4AⅢ,and BoGRP2A,in the yeast two-hybrid experiment. |
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