【作者】 李旺；

【导师】 陈玉林；

【作者基本信息】 西北农林科技大学 ， 动物营养与饲料科学， 2010， 博士

【摘要】 纤维素是世界上最丰富的可再生资源,最终需要在纤维素酶的作用下分解为葡萄糖才能应用在生产生活的各个领域。如何提高动物尤其是草食动物对粗纤维的利用率,一直是人们努力研究的问题,应用纤维素酶便是重要的手段之一。纤维素酶广泛存在于微生物、动物和植物体内,对纤维素具有特异性的降解作用。乳酸杆菌是一类广泛存在于自然界的有益菌,具有特异的粘膜黏附性。寄生在动物消化道的乳酸杆菌能够耐受消化道环境,对动物生长和免疫有促进作用。本研究以构建重组乳酸杆菌,使之既能表达纤维素酶又对动物有益生菌的作用为出发点;用雏鸡为动物模型,以建立从基因克隆、整合载体构建、宿主菌的筛选与重组到消化道的定植与表达这样一个完整的技术体系为主要研究目的,进行了如下试验。1.采用改良CMC筛选培养基,在广泛筛选的基础上,从秦岭羚牛(Budorcas Taxicolor Bedfordi)粪便中筛选到一株纤维素分解菌。通过形态学和16SrDNA保守序列分析的方法将其鉴定为枯草芽孢杆菌,并命名为Bacillus subtilis LN。用两对特异性引物从其基因组中扩增到2个纤维素酶基因,一个大小1.5kb,连续编码500个氨基酸,命名为Cel15;另一个大小741bp,连续编码247个氨基酸,命名为Cel73。序列分析表明,这2个纤维素酶基因均属于纤维素分解酶家族的成员,且均能够在大肠杆菌中进行表达,表达产物大小分别为55kDa和27kDa,产物大部分以包涵体形式存在。2.采用MRS筛选培养基从成年鸡的嗉囊、小肠、盲肠的内容物及其上壁上分离得到6株乳酸菌,这些菌株均为革兰氏阳性菌,除一株为球形外,其余均为不规则的杆状。通过16SrDNA保守序列分析将其鉴定为:1株嗜淀粉乳杆菌(Lactobacillus amylovorus SNY), 2株罗伊乳杆菌(Lactobacillus reuteri XNY,Lactobacillus reuteri MNY),1株卷曲乳杆菌(Lactobacillus crispatus SBY),1株短乳杆菌(Lactobacillus brevis XBY),1株肠球菌(Enterococcus hirae MBY)。并与NCBI核苷酸数据库中的9个乳酸杆菌的16SrDNA序列共同构建系统遗传进化树,结果验证了16SrDNA保守序列比对分析结果的正确。3.采用基因克隆等分子生物学技术构建了分别包含Cel15和Cel73基因的整合载体pLEM4153和pLEM4154。通过优化的转化方法,将整合载体整合到了鸡消化道野生型乳酸杆菌(Lactobacillus reuteri XNY)基因组中,并获得两个重组乳酸杆菌XNY4153和XNY4154。通过实时定量PCR(Real time PCR)的方法对Cel15和Cel73基因在重组乳酸杆菌中mRNA表达量进行了检测。Cel15基因的mRNA的相对表达量为18849.5,Cel73基因相对表达量为1388。测定重组乳酸杆菌改良培养基发酵液的纤维素酶活力,结果为重组乳酸杆菌XNY4153最高为0.158U/mL,重组乳酸杆菌XNY4154最高为0.15U/mL。4.将重组乳酸杆菌和鸡小肠上皮细胞共培养,发现重组乳酸杆菌在体外能够黏附于小肠上皮细胞上。通过0日龄口腔灌服将重组乳酸杆菌接种到鸡消化道,在之后的7日龄、14日龄、21日龄和30日龄的雏鸡嗉囊、小肠、盲肠部位分离微生物,通过PCR特异扩增报告基因检测重组乳酸杆菌的存在与否。结果表明,重组乳酸杆菌可以定植到雏鸡消化道的嗉囊、小肠和盲肠等部位,30日龄能够检测到报告基因。接种重组乳酸杆菌对蛋雏鸡的体增重有显著影响(P<0.01)。同时,对饲料粗纤维的消化率差异也较显著(P<0.01)。说明,重组乳酸杆菌能够在雏鸡的消化道定植和表达,表达的纤维素酶对饲料粗纤维有分解作用。通过上述试验构建了乳酸杆菌表达系统,重组菌成功在雏鸡消化道定植,并对生长性能和饲料粗纤维消化率有显著提高。更多还原

【Abstract】 Cellulose is the most abundant renewable resource in the world; it can use in many kinds of areas of people’s living and production only when it become glucose hydrolyzed by cellulase. How to improve the utilization rate of feed crude fiber of animals’especially ruminant anmimals is an important problem. Using cellulase is one of the methods of improving the utilization of feed crude fiber. Cellulase widely reside in microorganism, animals and plants, it can degrade cellulose specifically. Lactobacilli are kinds of probiotic widely reside in the nature world, and can adhere to the surface of mucous membranes specifically. The Lactobacilli stick to animal gut can tolerance the gut environment, and can promote the growth and immunity of animals. This research tries to construt a recombined Lactobacillus, and then uses it as a probiotic which can express cellulase. The main objective of this research is using chicken as model animal; establish a completed technique system from genes clone, integrative vector construct, host strain screen and recombine to recombined strain permanent plant and express in chicken gut.1. We isolated a cellulose degrade bacterium from the feces of Qinling Golden Takin(Budorcas Taxicolor Bedfordi) using improved CMC medium based on widely screen. We identified the bacterium belongs to the Bacillus using analysis of morphology and the conserved sequence 16SrDNA, and named it as Bacillus subtilis LN. Two cellulase genes were cloned from the bacterium chromosome DNA by two pairs of special primers. One is 1.5kb encoding 500 amino acid continuously, named Cel15; the other is 741bp, encoding 247 amino acid continuously, named Cel73. They are both the members of the cellulose hydrolysase family, and can be expressed in E.coli. The expression products are 55kDa and 27kDa respectively. Most of the products reside in the form of inclusion body.2. We isolated 6 Lactobacilli from crop, intestine and cecum content and wall of chick, using screen MRS medium. The Lactobacilli were all gram positive strains. They are all rhabditiform excepet one is globular. They then identified and named by 16SrDNA anylysis. The results are: 1 strain is Lactobacillus amylovorus SNY, 2 strains are Lactobacillus reuteri XNY and Lactobacillus reuteri MNY, 1 strain is Lactobacillus crispatus SBY, 1 strain is Lactobacillus brevis XBY, and 1 strain is Enterococcus hirae MBY. The results were reformed by constructing homology tree of these strains and 9 other strains of Lactobacilli from NCBI nucleotide data bank.3. Two integrative plasmids pLEM4153 and pLEM4154 which containing cellulase gene Cel15 and Cel73 respectively was constructed using molecular gene clone technology. The plasmids can integrate to wild type Lactobacillus reuteri XNY, by using an improved electro transformation protocol. And two recombined Lactobacillus reuteri XNY4153 and XNY4154 were screened. The two cellulase genes can express in recombined Lactobacilli detected by Real Time PCR. The two genes’mRNA relative expression amounts are: gene Cel15 is 18849.5 and the relative amount of gene Cel73 is 1388. The cellulase activity of fermentation broth of recombined Lactobacilli was determined, the results are: the highest cellulase activity of recombined Lactobacillus XNY4153 is 0.158U/mL, and the highest cellulase activity of Lactobacillus XNY4154 is 0.15U/mL.4. The recombined Lactobacilli can adhere to the enterocyte, after co-cultured the recombined Lactobacilli and enterocyte. The recombined Lactobacilli were inoculated to chicken gut in 0 day by drench from oral cavity. The cellulase genes were amplified by PCR in 7 days, 14 days 21 days and 30 days in the content of crop, small intestine and cecum of chicken. Planting recombined Lactobacilli can improve the bodyweights of chicken significantly(P<0.01), and at the same time, the digestibility of crude fiber was also influenced significantly by planting recombined Lactobacilli in chicken gut(P<0.01). This showed that the recombined Lactobacillus can permanent plant and express in chicken gut, the expressed cellulase can improve the grow performance and digestibility of feed crude fiber.We have constructed an expression system of Lactobacillus; permanent planted the recombined strain in chicken gut, and improved the growth profomance and the digestibility of feed crude fiber.更多还原