Screening And Identification Of High Effective Gossypol-degraded Strains And Study On The Differential Proteomics

Cottonseed meal (CSM) is an important plant protein resource. However, only a little of cottonseed meal is used as a protein resource in feed industry because of the existence of free gossypol (FG), which is an anti-nutritional factor and harmful to animals. Recently, several methods have been developed for removing FG from CSM, such as solvent extraction of FG, chemical treatment with ferrous sulfate, microbial fermentation, etc. These methods play an important role in detoxification of CSM. The method of microbial fermentation not only can detoxicate cottonseed meal, but also can improve the nutritional value of cottonseed protein. The advantages of fermentation detoxification of cottonseed meal have been recognized. But so far, the molecular mechanism of fermentation detoxification is not clear. In this study, two new gossypol-degraded strains were isolated from a cotton-planted soil, which could grow on solid medium containing gossypol. The strains were identified by morphological and molecular methods. Meanwhile, the growth characteristics of gossypol-degraded strains were preliminarily investigated by analysing their culture parameters. Additionally, the differential expression proteins of gossypol-degraded strains under different carbon source were analyzed by proteomics. The main research results were as follows:1. Two strains with high efficiency of gossypol degradation were screened from soil with gossypol as sole carbon source and named AN-1 and MM-2, respectively. The results of the morphological observation and electron microscopy showed that AN-1 and MM-2 were filamentous fungi. And then the two gossypol-degraded strains were identified by molecular biology. The results showed that:the 18S rDNA sequence of AN-1 was found to have 99%similary with those of Aspergillus niger isolates, indicating AN-1 was referred to be A. niger; the 18S rDNA sequence of MM-2 was found to share 98%similary with those of Fusarium isolates; indicating MM-2 was referred to be Fusarium sp.2. The isolated gossypol-degraded strains were cultured in different media to establish the optimum incubation conditions. The results of growth curvres of AN-1 and MM-2 both showed that medium containing glucose was better than gossypol and the best period used to be studied was 60-84 h incubation. The maximal biomass of both strains cultured by glucose and gossypol was at 30℃and growth in glucose was better than in gossypol. Cultured by glucose and gossypol respectively, the pH mutative curves of strains AN-1 and MM-2 were both downtrend, and glucose was more obviously than gossypol. Effects of various nitrogen sources on growth of gossypol-degraded strains showed similar trends. The results indicated that both AN-1 and MM-2 cultured by ammonium and nitrate were superior to that by yeast extract and urea, and the best nitrogen source was sodium nitrate. The tolerance of strain AN-1 cultured on solid media was analyzed under five different concentrations of gossypol (0.05%,0.1%,0.2%, 0.4%and 0.8%). The results showed that the gossypol-degraded strain AN-1 had strong tolerance of gossypol, and the optimal dosage of gossypol was 0.2%.3. The mycelial proteins of gossypol-degraded strain AN-1 cultured in different carbon sources for 72 h were extracted by TCA/acetone method. Some satisfied 2-DE maps were obtained. The protein spots concentrated in the range of 25.0-66.2 kDa and spreaded in different gradient of pI. The 2-DE gels analyzed by PDQuest software showed that average 478 spots in glucose gel,592 spots in gossypol gels. Glucose as the master gel, average 293 spots were matched in gossypol gel and 51 protein spots were differently expressed. Among the fifty-one differential protein spots,47 PMF maps were obtained by MALDI-TOF MS. The PMF maps were searched in Mascot database. Twenty proteins were preliminarily identified, including 1 kinesin,2 glycerol-3-phosphate dehydrogenases,1 citrate synthase,1 ubiquitin,1 unnamed protein and 14 hypothetical proteins。...