【作者】 张欣；

【导师】 杨谦；

【作者基本信息】 哈尔滨工业大学 ， 遗传学， 2007， 硕士

【摘要】 植物病害一直严重威胁着农业生产,生防微生物的应用是植物病虫害综合治理的重要手段,是保护生态环境,实现农业可持续发展的有力保证。角毛壳菌(Chaetomium cupreum)作为一种重要的生物防治真菌,多年来倍受研究者们的关注。分别由六种不同植物病原真菌细胞壁、胶体几丁质、粉末几丁质和羧甲基壳聚糖为诱导物,模拟角毛壳菌(C. cupreum)自然的重寄生过程,研究了不同时间和不同诱导物诱导角毛壳菌产几丁质酶的影响。结果表明,上述九种诱导物均可诱导角毛壳菌(C. cupreum)产生几丁质酶;由植物病原菌细胞壁诱导产生的酶活性总体比由几丁质及壳聚糖诱导产生的酶活性高,由立枯丝核菌(Rhizoctonia solani)诱导产生的酶活性较高,最高可达25.44 U/mL,而由大豆疫霉菌(Phytophthora sojae)诱导产生的酶活性较低,最高酶活性不超过1 U/mL;同时,不同诱导物诱导角毛壳菌(C. cupreum)产生几丁质酶的活性及酶活最高时期有所不同。由杨树叶枯病菌( Alternaria alternata )细胞壁、尖孢镰刀菌(Fusarium oxysparium)细胞壁、胶体几丁质和粉末几丁质诱导角毛壳菌(C. cupreum)产生的几丁质酶粗酶液的最适作用温度均为40℃;在50℃以下均具有较好的热稳定性;酶促反应的最适pH值均为5.0;在pH值4.0-7.0的范围内均能保持良好的pH稳定性。变性剂和蛋白酶抑制剂对几丁质酶活性也都有一定的抑制作用,其中EDTA的抑制作用最为明显,在10 mmol/L的浓度下,几丁质酶几乎丧其活性,EDTA对金属离子有鳌合作用,这也间接说明几丁质酶酶活性依赖于金属离子。将角毛壳菌(C. cupreum)chi58基因成功地连接到表达载体pYES2中,构建的酿酒酵母(S. scerevisiae)重组表达载体pYES2-chi58能够在胞外表达蛋白,在诱导培养60 h时几丁质酶的活性达到最高值0.11 U/mL。将角毛壳菌几丁质酶活性与酿酒酵母中chi58基因的表达活性进行对比,结果表明几丁质酶基因在角毛壳菌中的表达活性比在酿酒酵母中的表达活性高。几丁质酶在生物防治和处理几丁质废物方面具有巨大的应用潜力,已成为当前研究热点。充分研究和利用几丁质酶将会给生产生活带来巨大帮助。更多还原

【Abstract】 The application of biological control is an important method of the synthesis control to plant diseases and insect pests and environment protection. It is also the the advantageous guarantee to the sustainable agriculture development.Through imitation the natural superparasitism process of C. cupreum, during which the six different disease fungus cell wall, colloid chitin, powder chitin and carboxymethyl chitosan are taken as the inducer repectively, we research on the effect of different time, different inducement to the production of chitinase from C. cupreum. The result shows that all different kinds of fungal cell wall, chitin and chitosan can induce the C. cupreum to produce chitinase; the chitinase activity induced by the cell wall of pathogenic fungus is higer than that of chitosan; the chitinase activty, induced by the R. solani, is higher than others’, of which 25.44 U/mL. In contrast, the C. cupreum can not express the chitinase when iduced by P. sojae. Meanwhile, the peak activity and period of the chitinase are also different when it is induced by different inducement, which demonstrates that different inducement may induce different kinds of chitinase.The optimization temperature of chitinase induced by the cell-wall of X. oryzae, F. oxysparium, colloid chitin and powder chitin is 40℃; the chitinase has better thermal stability when the temperature is below 50℃; the optimiza-tion pH value of enzymatic reaction is pH5.0; it can keep good pH stability in the range of pH4.0-7.0. Denaturing agent and protease inhibitor have some depressant effect on the activity of chitinase, of which EDTA is most obvious. It also illustrates that the activity of chitinase depends on the metal ion.By the method of RT-PCR, we seprate the cDNA sequence of chi58 gene from the total RNA of C. cupreum and successfully link it with the expression vector pYES2 to construct the S. cerevisiae recombinatant expression vector pYES2-chi58. The recombinatant can express the protein in the ectocell which can reach the peak valuce 0.11 U/mL when induced for 60 h. Compared the chitinase activity of C. cupreum with that of chi58 gene in S. cerevisiae, the expression activity in C. cupreum is higher than that of chi58 gene in S. cerevisiae.Chitinase has the huge application potential in the biology controls and processes, and has become the current research focus. Researching and using chitinase thoroughly will give tremendous help to the people’s production and life.更多还原