【作者】 白晓青；

【导师】 袁耀武；

【作者基本信息】 河北农业大学 ， 微生物学， 2010， 硕士

【摘要】 原生质体融合技术是一种微生物育种方法,该方法简便、易行。利用该方法选育目的菌株的过程中仍然存在许多需要解决的问题。本试验以康氏木霉和酿酒酵母为出发菌株,对原生质体融合技术进行了研究,确定了纤维素酶高产菌株康氏木霉原生质体制备及再生的适宜条件,为该菌细胞融合和诱变育种提供适宜的真菌状态。同时对融合株的一些遗传性状进行了相关研究。该研究为微生物育种工作奠定了基础。本试验对影响康氏木霉原生质体形成和再生的各种因素(菌龄,蜗牛酶浓度,酶解温度和时间,渗透压稳定剂的成份和浓度)进行试验比较。结果表明,康氏木霉原生质体制备的最佳条件:菌龄为22 h,蜗牛酶浓度为2.0%,酶解温度为30℃,酶解时间为2.5 h,蜗牛酶溶液中的渗透压稳定剂为0.6 mol/L NaCl,在制备康氏木霉原生质体时观察到原生质体的释放方式主要有两种:顶端释放和段端位释放。本试验原生质体制备方法可以获得细胞融合和细胞突变诱导所需要的原生质体数量。本试验对康氏木霉原生质体的再生培养基进行了研究,确定了康氏木霉原生质体的最佳再生培养基为0.8 mol/L蔗糖YPD再生培养基,康氏木霉原生质体在0.8 mol/L蔗糖YPD再生培养基上平均再生菌落数为4.13×105个/mL。同时观察到了康氏木霉原生质体的一种再生方式。本试验采用抗药性遗传标记和热灭活两种方法作为融合菌株的筛选标记,确定了制霉菌素的标记浓度为750 U/mL,无水乙醇的标记浓度为23%。康氏木霉的热灭活温度为67℃,时间为10 min。本试验采用康氏木霉原生质体单亲热灭活(酿酒酵母采用抗药性遗传标记)和双亲均采用抗药性遗传标记两种方法共筛出246株融合菌株。本试验初筛采用TTC作为显色剂筛选出发酵葡萄糖能够产酒精的融合株,然后将此融合菌株在羧甲基纤维素钠培养基上进行复筛,筛选出分解葡萄糖产水解圈较大的融合株,TTC和羧甲基纤维素钠培养基能够筛选出既产酒精又产纤维素酶的融合株。将筛选出的融合株进行遗传稳定性试验最终得到4株稳定融合菌株。本试验最后对遗传性稳定的4株融合菌株进行了个体形态、生化特性、特定基因序列和可溶性蛋白等方面的遗传性质研究,研究发现:融合菌株B1、B2、B3确实进行了基因重组,但其酶活和产酒精能力都比亲本低。但是这些研究为原生质体融合育种工作提供了有益的参考。更多还原

【Abstract】 The protoplast fusion technology is a way of microbial breeding.This method is simple and easy.But there are still many questions about selecting objective bacterial strain to be solved by this method.This paper is focused on Trichoderma koningii and Saccharomyces cerevisiae,protoplast fusion techonology and inherited characteristics of fusant are analyzed.To provide a suitable fungal material for protoplast-fusion and mutation-breeding of Trichoderma koningii.This research work for microbial breeding laid a foundation.Conditions for Trichoderma koningii protoplast preparation and regeneration including mycelium growth time,the content of cellulose,temperature and time for reaction,various compounding of osmotic stabilizers were examined.The optimal conditions for protoplast preparation of Trichoderma koningii were as follows:22h mycelium growth time,the content of cellulase was 2.0%,2.5 h reaction at 30℃,the optimal osmotic stabilizer of cellulase was 0.6 mol/L NaCl.Microscopical observation indicated that the protoplasts of Trichoderma koningii were largely released from the apex and hypha segment.A satisfied concentration of Trichoderma koningii protoplasts could be achieved for the cell fusion and the cell mutation induced in this research.The regeneration culture medium was researched , determining the optimal regeneration culture medium for Trichoderma koningii protoplast was 0.8 mol/L sucrose YPD.The research showed that 4.13×105 regeneration colonies were in this regeneration culture medium . One modes forming irregular outshoot was found to regulate regeneration of Trichoderma koningii.Fusants were got by two methods.Drug resistance genetic marker and the single heated died parent were used for selection marker in this experiment.The nystatin concentration was 750 U/mL,The alcohol concentration was 23%,the single heated died parent of Trichoderma koningii protoplast was heated up to 67℃,10 min.The 246 fusants were selectd by the two methods.The fusants through the preliminary selection for fermenting glucose to the alcohol were selected by TTC.Then the fusants through continuous selection for breakdown of glucose were selected by sodium carboxymethyl cellulose culture medium.The fusants have the strong ability for production alcohol.The fusants have the highest enzymic activity strain by TTC and sodium carboxymethyl cellulose culture medium.The selected fusants were to be tested for genetic stability.The results were that the four fusants were the genetic stability fusants.Individual figure、biochemical characteristic、specific genetic sequence and soluble protein were studied by this experiment.The results were that the fusants for B1、B2、B3 and B4 proceeded gene recombination,but the ability for enzymic activity and production alcohol were lower than the parents.But this experiment provided the foundation for protoplast fusion breeding.更多还原