产纤维素酶真菌的基因组重组初探

【作者】 于航行；

【导师】 曲音波； 宋欣；

【作者基本信息】 山东大学 ， 微生物学， 2006， 硕士

【摘要】 纤维素是自然界中存在最广泛的一类碳水化合物，同时它也是地球上数量最大的再生资源。纤维素酶(cellulase)能将天然纤维素降解生成纤维素分子链、纤维二糖和葡萄糖。利用微生物生产的纤维素酶将其转化为人类急需的能源、食物和化工原料，对于人类社会解决环境污染、食物短缺和能源危机具有重大的现实意义。 分子育种技术(molecular breeding)继承和发展了杂交育种，引进了新的概念和方法，可以加速育种进度。本文采用基因组重排(genome shuffling)进行分子育种，对象是整个细胞，能弥补诱变育种的不足。把通过诱变育种改良获得的多种不同的突变株进行多次连续的原生质体融合，使得不同菌株来源的基因组能够得到充分的重组，高通量检出得到一批性能改善的重组子，再进入下一轮的基因组重排，可大大提高重组率。 本课题以纤维素酶生产菌株斜卧青霉(Penicillium decumbens)为出发菌株，通过原生质体的制备、原生质体反复融合，使细胞进行基因组重排，得到一系列与亲本相比具有优越性的重组子。将这些重组子和亲本菌株进行了形态学，产酶活力以及胞外全蛋白电泳图谱分析比较，证实了重组子和亲本的亲缘关系，以及融合过程中的染色体发生的交换分离。 首先以青霉菌株M114和Ju-A10为例，研究了青霉的菌丝及孢子原生质体的形成和再生条件。菌丝制备原生质体的条件为3mg／mL纤维素酶和3mg／mL蜗牛酶对生长32h的菌丝体进行处理，处理时间为1h。对于耐受性孢子，加大酶的用量为5mg／mL，孢子预先萌发8h，酶处理12h～18h，原生质体产率可达85％。原生质体的再生采用葡萄糖再生培养基效果最好，再生率可达81％。 在PEG的诱导下进行了M114和Ju-A10的原生质体融合，得到融合子Fu-3。表型上它分别具有两亲本的特征性状，菌丝形态如M114，分生孢子如Ju-A10；其CMCase酶活可以达到M114的2.38倍，Ju-A10的1.86更多还原

【Abstract】 Cellulose is the most abundant organic raw material in the world, and it is the only renewable resource that is available in large quantities. Cellulase can decompose the natural cellulose into cellulose molecular chain, cellobiose and glucose. It has great realistic meaning for human being to solve environment pollution, food shortage and energy crisis that using cellulase produced by microorganism transforms cellulose to energy, chemical and food.Molecular breeding inherited and improved crossbreeding; it accelerated the breeding-schedule by using new notion and method. The object of genome shuffling is the whole cell, it made cross breeding to mutation strain, and cause different strain’s genome to recombine completely, the chance of various forward mutation concordances to a recon improves. Simultaneity, accumulative detrimental mutation was replaced by the sequence of wild type strain. Hence, improved recon was detected by high flux, and then entered the next genome shuffling; the forward mutation rate can be enhanced greatly.Therefore, using the Penicillium decumbens as original strain, which produces cellulase, this research subject prepared protoplasts and made recursive fusion of them to fulfill the recombination among cells, and then we selected a series of hybrids with the better quality than the parental strains by genome shuffling. We contrast the hybrids with the parental strains from the morphologic characteristics, cellulase activity and extra-cellular protein electrophoresis strips, thus make sure the relative relationship between the hybrids and parental strains, and interpret the exchange and separation of chromosome in the course of fusion.Take the strain M114 and Ju-A10 as examples; we studied the preparation and regeneration using the hypha and spores of Penicillium. The更多还原