| Ribosome-inactivating proteins (RIPs) are a family of enzyme, which could remove a specific adenine A4324from the highly conserved, surface-exposed, α-sarcin/ricin loop in the28S rRNA of eukaryotic ribosomes. The depurination destroyed the interaction between elongation factors and ribosome, thereby resulting in inactivation of ribosomes and inhibition of protein synthesis. Studies show that RIPs not only have immunomodulatory,"clean-up" of bone marrow, antivirus, antimicrobial, antifungal, antimutagenic, antitumor, but also could effectively enhance the resistance of host plant to pathogens. RIPs are widely distributed in the plant kingdom, especially the Cucurbitaceae family including bitter gourd, sponge gourd and pumpkin. Alpha-Momordicin (α-MC), a RIP isolated from Momordica charantia L., could inhibit protein synthesis in rabbit reticulocyte lysate and wheat endosperm system. Studies on the enzamital activity were not complete, while its applications have not yet been fully developed. In this study, the highly purified α-MC was obtained from E.coli heterologous expression system coupled with Ni-NTA affinity chromatography. Researches on the enzymatic activities of α-MC were as followes:1. We have constructed the recombinant expression vector pET-MC and transformed it into E. coli Rosetta (DE3) pLysS strain. After optimizing the cultural condition by varying induction time (6-18h), IPTG concentration (0.5-1.5mM), culture temperature (22-37℃), and the cell concentration (0.6-1.0for OD600), the recombinant α-MC protein was mostly present in the soluble fraction. Subsequently, α-MC protein was recovered from the bacterial cells using Ni-NTA affinity chromatography. The protein concentration was estimated to be85mg/L with Bradford method. SDS-PAGE electrophoresis showed the94.7%purity and the recovery of73.9%. 2. Results showed that the RNA N-glycosidase activity was concentration-dependent. Only the28S rRNA was affected when being incubated with low concentrations of a-MC, releasing the typical "Endo’s fragment". With the increase of protein concentration, total rRNAs were completely degraded, as smear bands appearing on the agarose gel. The antifungal activity towards F. oxysporum and F. solan were tested from both molecular and cell levels. In addition, a-MC could also effectively inhibit the growth of P. aeruginosa.3. The DNase-like activity of a-MC towards plasmid was concentration-dependent, metal-dependent, temperature-sensitive, and pH-stable. Circular dichroism was used to detect the conformational change in plamid DNA. To explore the relationship between the DNase-like activity and polynucleotide:adenosine glycosidase activity, five amino acids critical for the polynucleotide:adenosine glycosidase activity were mutated to alanine, respectively. After prokaryotic expression and affinity purification, five homogenous mutant proteins were obtained. The DNase-like activity was completely lost in the Y93A mutant. However, the DNase-like activity was more or less affected in the other four mutants. We speculated that the polynucleotide: adenosine glycosidase activity and DNase-like activity might be implemented by distinct but related domains.4. In order to develop SSR markers linked with a-MC gene, the FIASCO technology was utilized to isolate microsatellites from bitter melon genomic DNA. In total,16loci showed polymorphic in36accessions collected from five populations (Guangxi, Hunan, Sichuan, Jiangxi, and Hubei). These loci contained three to eight alleles, with He and Ho ranging from0.325to0.867and from0.000to1.000, respectively. No significant linkage disequilibrium was found between these polymorphic loci. These markers could also amplify corresponding fragments in Cucurbita pepo L., Luffa cylindrical L., Lagenaria siceraria L., and Cucumis sativus L. These marks will facilitate the mapping of a-MC gene, and are also useful for genus classification, relation studies, as well as the molecular marker assisting breeding of bitter melon and other closely related species. |
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