【作者】 章英；

【导师】 龙超安；

【作者基本信息】 华中农业大学 ， 园艺学， 2012， 硕士

【摘要】 柠檬形克勒克酵母(Kloechera apiculata)菌株34-9是一株对柑橘采后真菌病害具有良好防治效果的生防酵母菌株。为了提高其生防效力,进一步改良酵母以利于商业化运用,本实验主要对柠檬形克勒克酵母的遗传转化方法进行了探索,包括醋酸锂法、电转化、原生质体法及其他转化方法。在确立合适转化方法的基础上,我们将克隆抗病相关基因并使之在柠檬形克勒克酵母中高水平表达。实验室已经研究得出柠檬形克勒克酵母分泌产生的主要抑菌物质是苯乙醇,能显著抑制青、绿霉病的发生。本实验克隆了苯乙醇合成的关键酶丙酮酸脱羧酶基因PDC并在毕赤酵母中表达验证其活性,为下一步试验PDC在菌株34-9中大量表达作准备,以最终提高其拮抗效力。主要实验结果如下：1.对潮霉素B敏感性检测确定使用100μg/mL潮霉素B作为菌株34-9抗性筛选浓度。将构建了同源臂的重组载体转化到野生毕赤酵母菌X-33得到稳定转化子,验证重组载体可以用于转化。2.采用醋酸锂法、原生质体转化等方法进行了转化实验,对细胞浓度、质粒线性化和非线性化浓度、转化试剂浓度、转化后培养时间等条件进行筛选,多次试验并未得到转化子。原生质体细胞膜外DNase酶活性高,但是限制DNase活性后转化也未得到转化子,表明DNase活性并不是限制转化的主要因素。3.进行了电转化方法试验,计算酵母在不同电压下的存活率,本实验选取1.5kV电压进行电击,并设置其他转化条件。采用电转化方法获得了少量转化子,转化效率低,初步确定转化条件为酵母浓度109CFU/mL,50μgDNA,电压1.5kV,电容25IIF,电阻200Ω。4.克隆丙酮酸脱羧酶PDC基因。基因全长为1695bp,翻译564个氨基酸。构建毕赤酵母表达载体pPICZaA-PDC,将重组质粒转化进X-33,得到的转化子能够在甲醇诱导下正常表达。更多还原

【Abstract】 Kloechera apiculata strains34-9is an effective biocontrol microorganism in resisting postharvest pathogens of citrus. Molecular improvement on the strain34-9is necessary to be carried out to enhance its biocontrol activity and commercial application. In this study, we mainly explored the methods of Kloechera apiculata transformation, including LiAc, electroporation, spheroplasting and other methods. Based on the right transform method, we intend to clone and highly express the disease resistance genes from Kloechera apiculata. Our previous work showed that2-phenylethanol is the main antifungal compound in34-9, which can control the blue and green mould of citrus. On this basis, we isolated key enzyme gene PDC of phenylethanol production and expressed in yeast Pichia pastoris. This experiment would provide a experimental base for large-scale expression of PDC in34-9and enhancing the antifungal effect. The main results are as follows:1. The sensitivity of34-9to different Hygromycin B concentrations was examined. We showed that Hygromycin B at100μg/mL could serve for the selection of transformants. The recombinant vector containing homologous arm was successfully transformed into the wild-type host strain X-33, implying that the recombinant plasmid can be used to transform into yeast.2. The transformation was carried out by the LiAc method, spheroplasting and other methods. The factors in different concentrations were screened, including yeast cells, undigested circular plasmid, linearized plasmid, transformation reagent and recovery time after transformation, etc. Although processes were repeated many times, colonies were not found on selection plates. We found that there was no transformant when the activity of protoplast-associated DNase was extremely strong as well as it was inactivated. The results suggested that the DNase was not the main inhibitor for transformation.3. In order to optimize electroporation, the cell viability was measured under different applied voltages. We found that the1.5kV was best for transformation. Considering the other factors, the conditions which is109cells/mL,1.5kV,50μg plasmid DNA,25μF and200Ω, was adopted in the study. Few transformants were observed though the efficiency was not high.4. A pyruvate devarboxylase PDC gene which is1695bp and encodes594amino acids was isolated from the yeast Kloechera apiculata. Then, the secretory recombinant vector was transformed into X-33strain. After induced by methanol, the postitive transformants expressed successfully and protein worked normally.更多还原