摘要
纤维是人类生存的第二大必需物质,伴随石油、森林和土地资源的短缺以及人类生活质量的提高,开发非棉、非木纤维资源迫在眉睫。
由于生物法提取草本纤维能有效降低传统方法和常规方法对环境造成的严重污染,提高产品质量,降低生产成本,因此该研究已经在国内外广泛开展起来。
构建细菌突变体是发现新基因、分析基因功能、了解某些生物机理的一个重要途径。本研究通过转座子Mini-Tn10对迄今报道的草本纤维提取效率最高的菌株胡萝卜软腐欧文氏杆菌(Erwinia carotovora)变异菌株CXJZU-120进行随机突变,获得5523个突变体。采用非纤维素降解实效法和水解圈法等进行功能性鉴定,筛选出3个非纤维素降解活性降低或丧失的突变体,这为深入研究非纤维素降解机理,寻找与非纤维素降解相关基因和构建新一代高效菌株奠定了基础。
以可水解魔芋葡甘露聚糖的枯草芽孢杆菌(Bacillus subtilis)BE-91为材料,运用PCR技术从该菌的基因组中扩增出β-甘露聚糖酶基因(β-manA)片段,经过克隆、测序、BLAST分析,证实由该DNA片段推导的编码蛋白具有β-甘露聚糖酶的保守结构域,是该家族成员,又经SDS-PAGE分析,在相对分子质量为35kD附近可见明显的蛋白表达带。将其与表达载体PET28a连接后获得的重组载体通过电击的方式转化到草本纤维提取高效菌种胡萝卜软腐欧文氏杆菌(E. carotovora)CXJZU-120中,经DNS法测定,该工程菌与出发菌株相比,β-甘露聚糖酶的活力提高了183-259%。其非纤维素物质降解的多项参数较原始菌株有明显提高。
Fiber is the second most necessary material for human being’s survival. with the shortages of oil, forest and land resource and the improvement of the quality of human’s life, it is urgent to develop non-cotton,and non-wood fiber resources.
Because herbaceous fiber extracted biologically is the developmental trend which can reduce the heavy pollution, upgrade the product quality and lower the production costs, This study has been carried out widely at home and abroad.
Construction of bacterial mutant is one of the most important way to discover new genes, analyze functions of genes and find out certain biological mechanism. In this research, 5523 mutants were obtained by Mini-Tn10 mediated insertional mutagenesis in CXJZU120, the mutant of Erwinia carotovora which is the most powerful strain reported for the bio-extration of herbaceous materials. Going with halo-producing assay we used the mutants to degrade non-cellulose and screened three non-cellulose degrading reduced or free mutants. It laid the foundation for studying the mechanism of non-cellulose degrading, searching the genes which are related to biological non-cellulose degrading and constructing a new generation of efficient strain.
A strain of Bacillus subtilisBE-91 which can hydrolyze the glucomannan of Amorphophallus was used as material to isolateβ-mannanase Gene, manA, by PCR, A DNA fragment was amplified and cloned from BE-91 genomic DNA. Sequence analysis and SDS-PAGE of the extracellular protein showed that this DNA fragment encoded a putative protein containing the conserved domain ofβ-mannanase, it belonged to the family of mannanase, and an expected protein about 35kD was found. The recombinant vector which came from the ligation of the fragment and expression vector PET28a was transformed into powerful strain of herbaceous bio-extraction, Erwinia carotovoraCXJZU-120 by electroporation. DNS showed that the mannanase activity was 183-259% higher than the original strain, and a number of parameters had markedly been improved compared with the original strain.
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