Cloning And Analysis Of AP-2 Complexes Beta1 And Cysteine Protease Gene In Euphorbia Helioscopia

Euphorbia helioscopia L. belongs to Euphorbia (Euphorbiaceae) is an annual or two year herbaceous species, which contains latex, and the whole plant is used medicinally. The latex is the main storage location of the medicinal components, and the latex is the protoplast of laticifers. Therefore, the research of laticifer development can provide theoretical basis for studying the synthesis, accumulation and transportation of main medicinal ingredients. Our research group found that protoplast degradation is the most critical event in the development of E. helioswpia laticifers by observing laticifer ultrastructures, latex proteomics results of E. helioswpia also showed that there are 10 proteins in the lysosomal pathway. As a result, in this paper, cysteine protease and AP-2 complex subunits beta 1 among these 10 proteins were choosed to carry out the gene cloning, bioinformatic analyses and prokaryotic expressions. The results lay the foundation for the further study of laticifers and provide theoretical basis for laticifer development and regulation. The main results were as follows:1. Two genes were cloned from E. helioscopia. The gene of cysteine protease was named as EhCP. The length of the sequence is 1271 bp. Its Genebank accession number is KP995938. The gene of AP-2 complex subunit β-1 was named as EhAP-2B1. The length of gene sequences is 3176 bp, Its Genebank accession number is KP995937.2. EhCP bioinformatic analyses showed that the predicted molecular weight of EhCP protein is 40.48 kDa, the theoretical isoelectric point is 5.21. It contains three active sites, and has a signal peptide belonging to transmembrane protein. The protein is mainly existed in extracellular, is also present in the endoplasm and lysosome. Homology of EhCP protein with cysteine protease of Ricinus communis, Jatropha curcas, Populus trichocarpa is 87%,88% and 87% respectively. Phylogenetic tree analysis showed that the CP of E. helioswpia has the highest identity and the closest genetic relationship with Populus euphratica, Ricinus communis, Jatropha curcas and Populus trichocarpa. The secondary structure prediction showed that the alpha helix occupies 35.73%, the extended strand occupies 21.33%, the beta turn occupies 10.25% and the random coil occupies 32.69% in the polypeptide chain.3. EhAP-2B1 bioinformatics analyses showed that the predicted molecular weight of EhAP-2Bl protein is 100.37 kDa, the theoretical isoelectric point is 5.10. The protein has three conservative areas, belongs to a non-transmembrane protein which is mainly located in the cytoplasm. The similarity of EhAP-2B1 protein with that of Jatropha curcas is 100%, with that of Theobroma cacao and Citrus sinensis is 99%. Phylogenetic analysis revealed that the protein of E. helioscopia AP-2 has the closest genetic relationship with Ricinus communis, and its relationship with Jatropha carcas belongs to parallel evolution. The secondary structure prediction showed that the alpha helix occupies 45.63%, the extended strand occupies 17.17%, the beta turn occupies 5.65% and the random coil occupies 31.56% in the polypeptide chain.4. Recombinant protein of EhCP and EhAP-2B1 was expressed by procaryotic expression system, their molecular weights are about 40 kDa and 33 kDa, which are consistent with the result predicted by software.