| Ribosome-inactivating proteins(RIPs)are toxin proteins that can inhibit protein synthesis.It is reported that RIPs in same species are polymorphism and different in enzymatic activity.RIPs purified from Bougainvillea.have highly DNase activity and the low nonspecific toxicity,showing more processing application in agriculture and medical field.Nevertheless,current research only focus on medical field,systematic research on polymorphism of Bougainvillea RIPs is lacking,and it is worth exploring whether different isoforms would be different in their active characteristics.In addition,seeking for new RIP is helpful to solve the defects of short period and unstable of RIP-anticancer medicine made from known isoform Bouganin.Genes need to be transcribed and translated to function,and gene polymorphism can attribute to selective polymorphism in transcription and translation.That is,biological polymorphisms are reflected in both transcription and translation levels.In consequence,to identify the polymorphism of the protein,this paper first identify from the level of transcription and protein based on former studies.Based on this,we further analyze these proteins' phylogenesis and structure by bioinformatic methods in order to try to explain the reason of polymorphism and the possible different functions among these isoforms to provide new insights into potential drug targets.The results are following:1.In the biological process from transcription to translation,Bougainvillea RIPs are polymorphic.In terms of transcription,RT-qPCR tested all known isoform genes and unknown isoforms named BI1-BI7 but the expression content of each isoform has inter-individual and seasonal differences.In terms of protein,MS results show that Bougainvillea RIPs express seasonally and variously,among which the isoforms expressed in spring are the most(9 species),followed by summer(8 species),then winter(7 species)while minimum in autumn(5 species).The experimental results of transcription and translation demonstrate that Bougainvillea RIPs has the characteristic of seasonal regulation.This characteristic may be related with the discrepancy of seasonal environments.Furthermore,the selective expression of Bougainvillea RIPs in the protein level indicates that not all the isoforms can be translated into mature proteins,this may be related to their involvement in the functional differences in resistance to environmental stress which means different isoforms may undertake different biological functions.2.Bioinformatic analysis of amino acid sequence of Bougainvillea RIPs shows:(1)Bougainvillea RIP has a molecular evolution process,and active sites like Trp 198,Asp113,Lys200,and Ser202 in known isoform Bouganin have changed in other isoforms during the evolution process;(2)The significant discrepancy of Bougainvillea RIPs mainly locates at both terminals of sequence,particularly at C-terminal;(3)The signal peptide,subcellular localization and post-translational modifications of Bougainvillea RIPs.Combining with former study results,we conclude that the reason for the polymorphism of Bougainvillea RIPs may be that the protein is encoded by multiple genes due to genetic processes such as gene duplication and mutation.In addition,protein structure and function are closely related,the differences of Bougainvillea RIPs in the aspects of key active sites,space structures and signal peptide,final subcellular locations possibly result in the different functions,and the isoform named BI6 may have significant difference in functions because of the observed discrepancy between BI6 and other isoforms.To sum up,this study was the first to reveal the polymorphism of Bougainvillea RIPs from transcription and protein levels,and obtain 7 unrecorded isoforms from the database.Deeper analysis of RIPs protein structure by molecular biological methods further enrich the molecular biological information of whole world RIPs.Finally,it also provide theoretical basis to explore the medical application of Bougainvillea RIPs. |
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