【作者】 陈刚；

【导师】 杨谦；

【作者基本信息】 哈尔滨工业大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 在农业生产上,植物病害的防治一直是一个重要的环节,尤其是对于由真菌引起的病害。多数病害的防治主要依赖于化学农药的使用,但该使用易对环境造成污染,危害人类牲畜的健康。所以,人们应该考虑应用生物防治方法来控制植物病害。其中球毛壳菌(Chaetomium globosum)是目前研究用于生物防治的有益真菌,它产生细胞壁水解酶如β-1,3-葡聚糖酶,细胞壁水解酶破坏植物病原真菌细胞壁,杀死植物病原真菌。因此,球毛壳菌(C. globosum)是生物防治分子机理研究和优良生物防治基因克隆的理想生物材料。为了利用重组β-1,3-葡聚糖酶生产生物农药,根据毕赤酵母(Pichia pastoris)容易培养、外源蛋白表达量高等特点,本研究采用GS115毕赤酵母分泌表达系统作为表达工具。首先利用生物信息学手段,即BLASTX相似性搜索从已构建的球毛壳菌EST库中筛选得到β-1,3-葡聚糖酶基因(glu)的5’和3’端,设计引物扩增出球毛壳菌β-1,3-葡聚糖酶基因,并提交GenBank,得到的序列号为FJ587923。然后用立枯丝核菌诱导球毛壳菌后提取球毛壳菌RNA,经反转录和PCR扩增后得到β-1,3-葡聚糖酶基因的cDNA序列。将经EcoR I和Not I双酶切的β-1,3-葡聚糖酶基因的cDNA和酵母表达载体pPIC9K进行体外连接,构建酵母重组表达载体,得到重组表达质粒pPIC9K-glu。将pPIC9K-glu和pPIC9K空质粒分别用限制性内切酶BspEⅠ线性化后,电转化GS115毕赤酵母感受态细胞,转化子经甲醇利用表型筛选和PCR检测筛选得到2株低效利用甲醇的阳性转化子。对这两株阳性转化子发酵后取上清进行SDS-PAGE检测和酶活测定得到1株高效表达β-1,3-葡聚糖酶的阳性转化子。对上述得到的转化子进行酶活测定与酶学特性研究,并对重组GLU的抑菌性进行了初步研究。发现该GS115转化子在第3天出现产酶高峰,重组GLU在温度为45℃时酶活性最高。根据该发酵条件,以昆布多糖为底物测得的重组GLU发酵液上清酶活为1.31U。另外,分别以尖孢镰刀菌(F. oxysporum)、核盘菌(S. sclerotiorum)、立枯丝核菌(R. solani)、稻瘟病菌(P. grisea)、叶枯病菌(S. tritici),杨树烂皮病菌(V. sordida)的细胞壁作为底物,测定重组GLU粗酶液的酶活性。结果表明,重组GLU对植物病原真菌尖孢镰刀菌(F. oxysporum)、立枯丝核菌(R. solani)、稻瘟病菌(P. grisea)比对核盘菌(S. sclerotiorum)、叶枯病菌(S. tritici)、杨树烂皮病菌(V. sordida)的细胞壁的酶活性要高,说明重组GLU对前三种植物病原真菌细胞壁的水解作用更明显,间接证明了重组GLU对尖孢镰刀菌(F. oxysporum)、立枯丝核菌(R. solani)、稻瘟病菌(P. grisea)具有更好的抑制作用。通过转化毕赤酵母GS115得到的重组GLU具有较高的酶活性,且对某些植物病原真菌有较明显的生物防治作用,所以该研究具有较高的理论意义和实际应用价值。更多还原

【Abstract】 In agricultural production, it is always important to control plant pathogens, especially pathogenic fungi. At present, mainly chemical pesticides are used in the control of most pathogenic fungi. However, the use of chemical pesticides can lead to pollution of the environment and damage the health of human beings as well as livestock. The use of biocontrol for the inhibition of plant pathogens should be taken into consideration. Chaetomium globosum produces some cell wall hydrolases includingβ-1, 3-glucanase which can damage the cell walls of plant pathogenic fungi. Therefore, the fungus can contribute to biocontrol. As a result, C. globosum can be used in the search of molecular mechanisms and novel genes which can be applied to biocontrol.In order to produce biopesticides from a recombinantβ-1, 3-glucanase, Pichia pastoris expression system was used in this research, taking advantage of its high expression of extrinsic proteins and ease of cultivation. Initially, BLASX was performed to search the 5’and 3’sequences ofβ-1,3-glucanase(glu) from the C. globosum EST database, which had already been constructed. Then, the novel gene glu was cloned from C. globosum by PCR with appropriate primers designed according to their EST sequences and the gene sequence was submitted to the GenBank database with the accession number FJ587923. Next, RNA was extracted from C. globosum induced by R. solani and the cDNA of glu was obtained after reverse transcription and PCR. After that, the recombinant plasmid pPIC9K-glu was constructed successfully by ligating the cDNA and the vector pPIC9K which were both restricted using EcoR I and Not I.Electrotransformation into P. pastoris GS115 was performed after the recombinant plasmid pPIC9K-glu and pPIC9K control were linearized with BspEⅠ. The tranformants were screened using clone PCR and two positive transformants were obtained. Then, SDS-PAGE and activity assay were conducted to obtain one transformant which could highly express the recombinantβ-1, 3-glucanase (GLU).The expressed GLU protein was characterized and its inhibition effect on six plant pathogenic fungi was studied. The result showed that the GLU was produced most abundantly until the 3rd day of induction and its optimal temperature was determined to be 45℃. Based on these conditions, the highest activity of GLU was 1.31U using laminarin as substrate. In addition, the activity of recombinant GLU was determined using the cell walls from F. oxysporum, R. solani, P. grisea, S. sclerotiorum, S. tritici and V. sordida as substrates respectively. The results showed that the activities were higher using the cell walls from F. oxysporum, R. solani, P. grisea as substrates, whilst they were lower using the cell walls from S. sclerotiorum, S. tritici, V. sordida as substrates. It demonstrates that GLU can more drastically hydrolyze the cell walls from F. oxysporum, R. solani and P. grisea, and a conclusion can be drawn that GLU can inhibit the growth of F. oxysporum, R. solani and P. grisea more effectively.In conclusion, the recombinantβ-1, 3-glucanase, which was produced by the transformed P. pastoris GS115, showed high activity. And it could inhibit some plant pathogenic fungi obviously. Therefore, this study can contribute to biocontrol both theoretically and practically.更多还原