【作者】 杨文静；

【导师】 刘朝良；

【作者基本信息】 安徽农业大学 ， 生物物理学， 2011， 硕士

【摘要】 家蚕是产生较大经济效益和社会效益的重要经济昆虫，家蚕肠道微生物对其生长发育及微生态平衡具有重要作用。目前国内外关于家蚕肠道微生物的研究报道，多集中于肠道微生态系统结构、菌群致病性等方面，而关于家蚕肠道菌应用于产酶益生菌的研究较少。肠道产淀粉酶细菌，可帮助宿主更好地消化淀粉等营养物质，此外该类菌还有许多其它的益生功能。近些年来，关于益生菌在动物微生态制剂方面的应用，多集中于水产类及家禽生产中，而蚕用益生菌的研究未见报道，蚕业生产过程中一直存在着叶丝转化率较低，蚕病发生率高等问题，因而研究蚕肠道菌及其产酶并对产酶基因进行原核表达分析，在改善蚕肠微生态环境及蚕人工饲料开发上有一定意义。本文从大造品种家蚕5龄幼虫肠道中通过传统培养法分离纯化出部分可培养细菌，通过含淀粉的牛肉膏蛋白胨培养基筛选得到其中两株产淀粉酶菌株，用细菌扩增通用引物分别对产酶菌进行16SrDNA序列扩增并克隆测序，将所得测序结果与GenBank已有序列进行比对，该两株菌均与蜡样芽孢杆菌序列有99%的同源性，结合菌落形态学观察及菌体细胞革兰氏染色涂片显微镜镜检分析结果，鉴定所得菌株为芽孢杆菌属蜡样芽孢杆菌，DNS法测定菌株所产淀粉酶酶活，结果显示，该家蚕肠源性细菌所产酶具一定酶活力，且菌株的16SrDNA序列也与已有报导的蜡样芽孢杆菌序列有一定差异，因而具有一定的研究意义。鉴定得知分离产酶菌种属后，根据已解析的蜡样芽孢杆菌α-淀粉酶(Ec.3.2.1.1)基因设计引物，从该两株细菌的基因组DNA中分别扩增出了产淀粉酶基因片段，经克隆、测序和分析获得了家蚕肠源性蜡样芽孢杆菌淀粉酶基因的完整编码区ORF框序列，产酶基因扩增分别得到3414、3378个碱基序列，均包括完整的编码区1761bp，可编码586个氨基酸，比对分析属于α-淀粉酶超级家族，但这两株菌的产酶基因序列存在一定差异，其中，DZ-a号菌793位碱基为T，而DZ-h为C，DZ-a号菌1381位碱基为A， DZ-h为G，对应的氨基酸序列DZ-a号菌265位氨基酸为S，DZ-h为P，DZ-a号菌461位氨基酸为I，而DZ-h为V。将DZ-a号菌α-淀粉酶基因在大肠杆菌表达系统进行原核表达，得到分子量为66kD左右的目的蛋白，与DNAStar软件预测产物蛋白大小相符合。根据该编码区序列设计含酶切位点的引物重新扩增获得目的基因序列，连接到表达载体pET-28a(+)构建了该产酶基因的原核表达载体，经酶切鉴定后将该表达载体转化到含稀有密码子编码氨基酸的大肠杆菌宿主细胞Transetta（DE3）中，用IPTG进行了不同浓度的诱导表达，经SDS-PAGE电泳检测，表明被诱导的目的蛋白均得到了稳定的表达，这为以后对该菌株淀粉酶基因的真核表达及其功能的进一步研究奠定基础。更多还原

【Abstract】 Silkworm is an important economic insect which has a great economic and social benefit.Microorganisms in silkworm gut have an important function for their growth and micro-ecological balance.Most reports about silkworm gut bacteria focused on the structure of their micro-ecosystem and nosogenesis while less reports about their application in enzyme-producing bacteria at present.Amylase producing intestinal bacteria can help the host digest starch or other nutrients more effectively.And these bacteria have some other probiotic functions.In recent years,probiotics application in animal microbial preparation concentrated in the production of aquatic and poultry production.Yet few study was about silkworm probiotics.Problems on low leave silk changing rate and high silkworm disease incidence in Sericulture always existed,so make research about silkworm bacteria and their enzyme production ability then make a prokaryotic gene expression analysis about enzyme gene may have some significance in improving the silkworm gut microenvironment and in development of silkworm artificial diet.Some culturable bacteria in larval guts of the Bombyx mori at5th year phase were isolated and purified by traditional method.Two amylase-producing strains of them was screened though beef extract peptone starch medium.Enzyme-producing bacteria’s16SrDNA sequences were amplified and cloned with bacteria identification universal primers respectively.Blasted these cloning sequences with sequences existed in GenBank,and the two strains both have99%homology with Bacillus cereus.Identified these strains we got as Bacillus cereus combining with observation of their colony shape and observation of their individual shape by microscope.Determining strains’ amylase activity by DNS method.Results showed,enzyme produced by the silkworm gut bacteria had a certain activity,and strains’16SrDNA sequences were also different from sequences existed in GenBank,and thus has some significance.Primers of α-amylase genes(Ec.3.2.1.1) were designed according to known genes after identification of isolated strains.Amylase gene sequences were amplified from genomic DNA of the two strains.Complete open reading frames sequences were got after cloning,sequencing and taking analysis of the Bacillus cereus.Sequences analysis of the amylase genes showed that the cloned sequences of the two strains were3414bp and3378bp respectively.They both contained complete open reading frames1761bp encoding586amino acid residues.Isolated these amino acid residues as α-amylase superfamily,but sequences of the two strains were diffieret.Among them,the793th base of the strain DZ-a was T,while the strain DZ-h was C.The1381th base of the strain DZ-a was A,while the strain DZ-h was G.So the relevant amino acid of them,the265th aa of the strain DZ-a was S,while of the strain DZ-h was P.The461th aa of the strain DZ-a was I,while of the strain DZ-h was V.α-amylase gene of the strain DZ-a were expressed in Escherichia coli prokaryotic expression systems.The target protein about66kD was got,and this was matching the size of its predicted protein product by DNAStar software.Primers containing enzyme restriction sites according to the coding sequence were designed to amplify target gene sequences.Then linked them to the expression vector pET-28a (+) and constructed a prokaryotic expression vector.The vector was digested and identified then was transformed into the Escherichia coli host cell Transetta (DE3) which containing infrequent codons.Induced their expression by different concentrations of IPTG.SDS-PAGE electrophoresis detection indicated that the target protein all had a stable expression.These results will provide the foundation for further eukaryotic expression of the strains’ amylase gene and for further function study of them.更多还原