【作者】 石君；

【导师】 刘正初；

【作者基本信息】 中国农业科学院 ， 微生物学， 2008， 硕士

【摘要】 木聚糖酶在饲料、制浆造纸、食品、医药、能源工业领域具有广阔的应用前景。随着分子生物学、结构生物学及蛋白质工程的发展,木聚糖酶结构和功能的研究不断取得新的突破;有关木聚糖酶基因的研究也取得了很大进展,不仅克隆出了许多不同来源的木聚糖酶基因,而且初步明确了木聚糖酶基因在同源和异源宿主中的表达和分泌机制。中国农业科学院麻类研究所刘正初研究员等在国内外率先采用基因工程手段,将编号为ly菌株的质粒转化到Erwinia carotovora中,获得一系列欧文氏杆菌变异菌株。本研究在前期工作基础上,筛选到了高产木聚糖酶的菌株欧文氏杆菌CXJZ11-01,构建了其功能基因组文库,从中克隆出木聚糖酶基因,明确了该基因的分子结构,为构建木聚糖酶基因高效表达体系和获得草本纤维精制高效工程菌株等深入研究奠定了基础:1、采用产木聚糖酶菌株选择培养基及DNS检测方法,从300多个欧文氏杆菌变异菌株中筛选出木聚糖酶高产菌株CXJZ11-01。复筛菌株在筛选平板上的透明圈大小和测定其发酵液木聚糖酶活性的结果一致表明,CXJZ11-01的木聚糖酶活性>>CXJZ11-03>CXJZ11-02>T85-1。2、通过提取CXJZ11-01菌株的DNA和优化酶切、连接、转化技术体系,构建了草本纤维提取高效菌种CXJZ11-01的木聚糖酶功能基因组文库。直接回收大小在1-5kb之间的酶切DNA片段进行转化,采用透明圈法筛选获得了2个木聚糖酶基因阳性克隆。3、从阳性克隆中提取质粒,采用酶切和PCR方法对酶切片段进行鉴定、序列测定,获得了木聚糖酶基因克隆,该基因已经登陆GenBank,登陆号为EU233656。对该木聚糖酶基因进行生物信息学分析与表达研究结果证明,该基因片断长度为708bp,包括一个长度为642bp的完整开放阅读框,编码213个氨基酸,分子量为23.36KDa。该基因翻译的213个氨基酸可能有29个氨基酸的信号肽序列,编码的蛋白为胞外蛋白,属于糖苷水解酶第11类。更多还原

【Abstract】 Xylanases have wide commercial applications in industrial processes,such as feed ,paper and pulp,foodstuff and energy industry. With the development and application of the new technologies , such as molecular biology , structural biology and protein engineering , many progresses have been made in the research of structures and functions of xylanase. With the development and application of the new technologies ,many progresses have been made in the study of xylanase gene , including the molecular cloning ,the expression and secretion in homologous and heterogenous hosts , the structure and the function of xylanases and in the reconstruction of the properties of xylanases for industrial production. Research Fellow Liu Zhengchu , Institute of Bast Fiber Crops in CAAS, has taken the lead of transforming the plasmid in strain ly into the Erwinia Carotovora and obtaining a series of variant strains by means of genetic engineering. On the basis of preparatory work, we have screened the high-yield of xylanase strain Erwinia Carotovora CXJ11-01. the genomic library of Erwinia Carotovora CXJ11-01 was constructed, and the xylanase gene was cloned from the library. The xylanase gene sequencing and analysis was also completed. This study will lay a foundation of further research on the construction of xylanase high efficiency expression system and the obtaining of efficient herbaceous extraction gene engineering strains:1 By using selective culture media and DNS detection method, we screened xylanase high yielding strain CXJ11-01 from more than 300 Erwinia Carotovora variant strains. The results from comparing the dimension of the transparent zones of the secondary screening strains on the plates and determining the enzyme activity of the fermentation broth all indicated that the enzyme activity for xylanase is: CXJZ11-01>>CXJZ11-03>CXJZ11-02>T85-1.2 By using shot-gun method,the genomic DNA library of the efficient herbaceous extraction strain E.carotovora CXJZ11-01 was constructed.Using the PCR method to the positive clones, it is indicated that they included the identical xylanase genes.The xylanases gene was successfully cloned,analyzad and expressed.3 the genomic DNA library of the efficient herbaceous extraction strain E.carotovora CXJZ11-01 was constructed and its xylanases gene was successfully cloned.The gene is registered at GenBank numbered EU233656.The sequence length of the xylanases gene is 708bp,including a 642bpORF which encodes for 213AA of molecular weight of 23.36KDa.It’s an extracellular enzyme classified as hydrolase 11.更多还原