| Lignocellulose is the most widely distributed and the most abundant renewable resources in nature. Conversion of cellulose and hemicellulose to fermentation sugar effectively isthe key process in biofuels productionby using lignocellulose. At present, the hemicellulase activity is low in most of the commercial cellulase products and is not enough in converting the hemicellulose components in the lignocellulose material. The hemicellulose, which is not degraded in the raw material of lignocellulose, can hinder the degradation of cellulose. Therefore, the addition of hemicellulase into the cellulase system can greatly improve the efficiency of converting cellulosic materials into fermented sugar.For a long time, the difficulty in highly expressing cellulose degradation enzymes has been a bottleneck in the study of its structure and characterizations.Traditional bacterial and yeast expression systems have their own defects in the expression of filamentous fungi proteins because the expressed proteins have to be modified before they are secreted extracellularly. The protein expression systems in filamentous fungi have their irreplaceable advantages especially in expressing the proteins from filamentous fungi, therefore, filamentous fungi are the ideal expression hosts for the expression of filamentous fungi-derived lignocellulose degrading enzymes.RNA interference (RNAi), which can turn down gene expression effectively and specifically, has showed great potential in gene function research and gene modification. In filamentous fungi, the traditional gene knockout methods can not work well because the polykaryocytosis and non-homologous recombination were usually occurs in filamentous fungi, especially some lethal genes refer to cell vital movement, thus, knocking down those genes partially is a preferable way to study their functions in vivo. RNAi technology is more suitable in studying those significant but lethal genes when compared to gene knock-out which are more difficult, complicated and time consuming.Therefore, RNAi technology is of great significance for the study of the functional genes of filamentous fungi and the transformation of strains.The main contents and results of this paper are as follows:1. Overexpression of 10 hemicellulasessin P. oxalicum A15A and saccharifying ability of recombination strains.P. oxalicum Δ15A was used as the host for the overexpression of 10 hemicellulasess from P. oxalicum. These 10 hemicellulases in the P. oxalicum 114-2 was not secreted or low amount. SDS-PAGE showed that all of the 10 hemicellulases were obtained respectively in P. oxalicum Δ15A.Then the corncob residue (CCR) and alkali pretreated corn stalk (ACS) were used as substrates to evaluate the saccharifying abilities of the recombinant cellulase producted by the recombination strain. The results showed that except Aga27A, the other 9 hemicellulases could improve the ability of the enzyme to degrade the CCR or ACS and the effect of XynlOB on CCR was more obvious while the effect of Xyn10B on ACS was more obvious.2. The expression of hemicellulases in P. oxalicum A11Δ.In order to further study the properties and applications of hemicellulasess that have been selected out, we used P. oxalicum A11Δ as the host to express the homologous hemicellulases, and purified the hemicellulases. SDS-PAGE result showed that hemicellulases were purified to be homogenousand protein concentration was very high.In the study of enzymatic properties, the substrate spectrum of the hemicellulases were analyzed and the results demonstrated that three of the hemicellulases are xylanases and one isferuloyl esterase:We analyzed the effects of the temperature and pH on the 4 hemicellulases activity, which showed the Xyn10B optimum temperature was 40℃, and Xyn30A Xyn10C optimum temperature was 60 ℃; Xyn30A optimum pH was 5,and the Xyn10B and Xyn10C optimum pH was 6; thermal stability of Xyn10B was relatively poor. The Fae1A optimum temperature was 40℃, and its optimal pH was 7; in addition, the experimental results showed that the thermal stability of FaelA was pretty good.In order to analyze the effect of the hemicellulases addition to cellulose system on degradation of cellulose materials, the four hemicellulaseswere respectively added to commercial cellulase and fermentation broth of Trichoderma reesei QM9414to investigated their synergistic effects on the degradation ofe CCR and ACS. The results showed that Xyn10B, Xyn30A, Abf43C, Fae1A and Axe1A could improve the degradation efficiency of commercial cellulose on CCR. The synergistic effects of Axe1A and Fae1A were more significant, Xyn10B, Xyn10C, Axe1A as well as Xyn30A could improve the degradation onACS. Xyn10B, Xyn10C and Xyn30A could improve the ability of Trichoderma reesei QM9414 crude enzyme degradation onCCR, the effects of Axe1A, Xyn10B, Xyn10C and Xyn30A on ACSwere more obvious. The results showed that xylanase and esterase could promote the biological conversion of cellulose materials.3. Construction of RNA interference vector for PenicilliumA RNA interference vector pHX-RNAi that carried a hygromycin resistance cassette and a transcriptional unit for hairpin RNA expression was constructed. A gene amy15A which encoded the major amylase in a high cellulase producer strain P. oxalicum 114-2 was targetedly knocked down. The results of SDS-PAGE and RT-qPCR showed that were both dramatically down-regulated at transcription and secretion levels of Amy 15A, this implied that RNAi played a significant role in regulation of gene expression post-transcriptionally. All of the hygromycin-resistant transformants with the silenced sequences showed high degree of silencing in expression of amy15A. Then amy15A and cel7A-2 were connected to the pHX-RNAi. The results of enzyme activity assay, SDS-PAGE and RT-qPCR analysis showed that the expression of amy15A and cel7A-2 were severely inhibited, which indicated that multiple genes could be silenced by only one step. The gene silencing mothed was very promising in genetic modification of filamentous fungi. Since RNAi mechanism is conservative, the high efficient RNAi vector pHX-RNAi will be universal and can be applied in other filamentous fungi to silence genes. |
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