【作者】 李方正；

【导师】 赵宏坤；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 纤维素酶作为饲料添加剂,可提高饲料利用率和粗饲料的营养价值,降低饲料成本,在促进动物生长、减少粪便排放、改善生态环境和动物疾病防治等方面效果明显,同时通过纤维素高效降解菌发酵处理秸秆,可以合成含有丰富蛋白质的菌体细胞,这些分解产物和菌体亦可用于单细胞蛋白饲料。乳酸菌是人和动物肠道内的常见细菌,被公认为安全级(Generally Recognized As Safe, GRAS)微生物。它作为一种益生菌,具有维持肠道菌群微生态平衡、可激活机体粘膜免疫反应、抑制肠道内病原微生物的生长、增强动物机体免疫力等多种生物学功能。另一方面,基因工程乳酸菌可以集益生和外源基因表达的双重功能于一身,在功能食品、医疗保健、人工口服疫苗等领域的应用均具有诱人的前景。本实验从奶牛、山羊瘤胃内容物以及腐烂饲草中通过羧甲基纤维素钠(CMC-Na)平板选择培养刚果红染色法初步筛选到能够产生明显透明圈的细菌菌株18株、真菌菌株33株,通过测定纤维素酶活性进行复筛,分离到高效纤维素降解真菌和细菌各一株作为后续实验研究对象,通过克隆真菌NL-3的18S rDNA序列,并以其同源性为基础构建相关种属的系统发育树。结果表明, 18S rDNA序列全长504bp, Blast显示该菌株与青霉菌属同源; NL-3菌株在进化关系上也与青霉菌属(Penicillium)聚成一族。特别是与Penicillium decumbens strain JU-A10的同源性最高,其序列相似性达99%。结合菌株形态学与18S rDNA基因序列分析对分离菌株进行鉴定,初步鉴定为青霉属的斜卧青霉(Penicillium decumbens)。同时克隆了细菌N9的16S rDNA序列。测序分析表明,其16S rDAN序列全长1514bp。BLAST显示该菌株与枯草芽孢杆菌(Bacillus subtilis)同源。以同源性为基础构建了相关种属的系统发育树,该菌株在进化关系上与枯草芽孢杆菌聚成一族;结合生理生化特征及16S rDNA分子鉴定,将其鉴定为枯草芽孢杆菌。根据GenBank上已发表的枯草芽孢杆菌内切纤维素酶(eg)基因序列,针对pMG36e质粒的多克隆位点设计引物,以枯草芽孢杆菌N9的基因组DNA为模板,扩增eg基因序列。该基因序列全长1518bp,通过序列比对分析,eg与已报道的其它来源的内切纤维素酶的序列相似性最高在92%。并将其亚克隆至乳酸乳球菌表达质粒pMG36e中,构建重组乳酸乳球菌表达质粒pMG36e-eg。通过电转化方法将重组质粒转化至乳酸乳球菌MG1614感受态细胞,成功构建了产内切纤维素酶的基因工程乳酸菌。将此重组转化子点接至含CMC的M17平板上能够产生透明圈,收集表达产物后通过进行SDS-PAGE电泳、Western blot检测到内切纤维素酶分子量约54KD,与预期结果相吻合。本研究筛选到多株高效纤维素降解菌,并克隆了内切纤维素酶基因(eg),构建乳酸菌表达载体pMG36e-eg,构建了重组内切纤维素酶基因工程乳酸菌。以期利用乳酸菌表达系统得到高活性纤维素酶以及开发出产内切纤维素酶的多功能微生态制剂。更多还原

【Abstract】 Cellulose as a feed additive can improve feed utilization rate and nutritional value of fodder and reduce feed costs. It is obvious in the promotion of animal growth, reduction of dejecta, the ecological environment improve and animal disease control. Meanwhile, through the fermentation of cellulose-degrading strains bacterial cells rich in protein are synthesized which can be used for single cell protein feed.Lactococcus lactis is the beneficial bacteria in annimal and human being’s gut, it is generally acknowledged as a security microorganism, on the one hand, it could maintain the ecological balance in the gut, stimulate the mucous membrane immune response of the organism, on the other hand, the recombinant gene Lactobacillus could be useful an resource for the development of functional foods, medical care and oral vaccine.Congo-red staining method was used to isolated 33 fugues and 18 bacteria strains of cellulose-decomposing from the rumen of the cows and sheep and rotten fodder. According to the detection of cellulase activity,one fungi and one bacteria strain with high efficient cellulose decomposing were isolated. The results showed that the complete 18S rDNA gene sequence was 504 bp. A phylogenetic tree was constructed by comparing the 18S rDNA sequence with other relative fungus species in the GenBank database. In the phylogenetic tree the strain NL-3, Penicillium decumbens strain JU-A10 constitute a branch with the similarity value 99%. According to morphologica characteristics and phylogenetic analysis, the strain NL-3 was identified as Penicillium decumbens. A phylogenetic tree was constructed by comparing the 16S rDAN sequence of the strain with other relative bacteria species in the GenBank database. According to Physiological and biochemical characteristics and phylogenetic analysis, the strain N9 was identified as Bacillus subtilis.According to the sequence of eg published on GenBank, eg gene was PCR amplified from N9 genome DNA by primer designed for Multiple cloning site of pMG36e. The complete gene sequence was 1518bp. By sequence analysis, the similarity value betwwen eg and other endo-cellulase was 92%. Recombinant Lactococcus lactis plasmid vectors pMG36e was constructed by subcloning eg gene. Recombinant genetic engineering Lactococcus lactis producing endoglucanases was constructed by transforming recombinant plasmid into competent cell of Lactococcus lactis MG1614. The recombinant transformants vaccinated to the M17 plates containing CMC could produce degradation halo. The sizes of expressed recombinant proteins was 54 KD by SD-PAGE analysis and Western blot analysis.Screening of high effective cellulose degradation strains, cloning of eg gene, constructing of Lactobacillus expression vector pMG36e-eg and recombinant genetic engineering Lactococcus lactis producing endoglucanases were completed in the study. It is expected that highly active cellulase are produced to use Lactobacillus expression system , and multi-functional microecological products producing endo cellulose are developed.更多还原