【作者】 任雪艳；

【导师】 郑晓冬；

【作者基本信息】 浙江大学 ， 食品科学与工程， 2012， 博士

【摘要】 由真菌病原菌引起的果实采后病害对人们造成了严重的经济损失。用拮抗酵母取代化学杀菌剂,或降低化学杀菌剂的使用量(即生物防治BCPD),具有良好的应用前景,但是普通拮抗菌很难达到化学杀菌剂的杀菌效果,因而筛选新型拮抗菌或提高原有拮抗菌拮抗效力成为了该领域的研究热点。通过基因工程技术改造拮抗酵母从而提高其生防效果是果蔬采后病害生物防治的新方法。本文探讨了将抗菌肽基因转移到酵母中表达以提高其生防效果的方法,成功的将外源抗菌肽基因CecropinA和PsdI在毕赤酵母中进行表达并对重组酵母GS115/CEC、GS115/PSD的安全性及对果蔬采后的主要致病菌和果蔬真菌病原菌引起的腐烂的抑制效果进行了研究；探讨了该酵母与外源物质结合使用对果蔬采后病害抑制效果的影响；初步研究了该酵母对果蔬致病菌的抑制机理及对果蔬贮藏后品质的影响,其主要研究结果如下：(1)根据已报道基因序列合成抗菌肽Cecropin A和PsdI基因片段,并将其连接到PGEM-T载体上。在此基础上将目的基因插入酵母表达载体pPIC9K,构建毕赤酵母表达载体。将其线性化后,用电转化法导入Pichia Pastoris GS115中。采用G418进行梯度筛选高抗性转化子。以甲醇作为诱导物,取发酵培养3d后的发酵上清液,进行Tricine-SDS-PAGE验证了基因表达的正确性。并且经接种培养、诱导发酵及PCR鉴定等验证了重组酵母具有良好的稳定性。(2)通过对重组毕赤酵母GS115/CEC、GS115/PSD发酵过程的两个阶段发酵条件的优化实验,得出生长阶段优化后的发酵工艺为：甘油2%,硫酸铵2%,pH=6.5-7.5,30℃条件下培养72h。外源基因诱导表达阶段优化后的发酵工艺为：甲醇1%,硫酸铵2%,pH=7.0,30℃条件下培养72h。采用正交实验来优化毕赤酵母的发酵条件,其影响发酵的主要因素及影响的大小依次为：温度>甲醇浓度>发酵天数>溶液pH值。最终得到重组毕赤酵母发酵过程中各项影响因素的优化值,即pH5.5,甲醇浓度为1%,温度为30℃,时间为4d,在优化后的发酵条件下,重组酵母GS115/CEC、GS115/PSD表达的外源蛋白含量平均可达15.1及11.3 mg/L。(3)利用重组酵母对4种果实的4种主要采后致病菌进行了体外及体内生防实验：平板抑菌实验及液体抑菌实验均表明重组酵母在体外对病原菌有广谱抑菌活性；实验表明该重组酵母可显著抑制链格孢、灰葡萄孢在番茄果实上的侵染；抑制扩展青霉引起的苹果及梨的腐烂；抑制灰葡萄孢引起的梨和苹果腐烂。同时实验表明,重组酵母对病原真菌的抑制效力与其细胞悬浮液的浓度有关,浓度越高,对致病菌的抑制作用也越强。(4)对重组酵母抑菌机理的研究结果表明,抑菌机制除体现了传统的营养与空间的竞争外,采用扫描电镜、透射电镜观察发现表达抗菌肽基因的重组酵母处理的真菌外部形态和内部结构都发生了很大的变化,出现了畸形,破裂,断残等现象。证实了其对真菌的膜结构及细胞内部结构的损坏作用。(5)在以上工作的基础上,进一步从分子水平研究了表达抗菌肽基因的重组酵母对真菌的作用机制。凝胶阻滞实验证明,抗菌肽能与真菌的RNA结合,影响了它们在电泳中的迁移率,同时还能使真菌的DNA及RNA降解,因而真菌细胞的代谢过程受到破坏,最终导致了真菌的死亡。此外,重组酵母能诱导与果实抗性相关的PR蛋白过量表达,因此诱导果实的自身抗性可能也是抑菌机制的一部分。(6)重组酵母能和外源物质相结合,显著增强对果实采后病害的防治效果：重组酵母和适当浓度金属盐类(氯化钙)、碳酸盐及乙醇结合使用,能降低果实的腐烂率；采后水果腐烂率显著低于1×108 cells/mL GS15/CEC单独处理的水平。(7)用重组酵母GS115/CEC、GS115/PSD的干粉制剂进行了食品级小鼠急性经口毒性实验,初步确定该制剂属于实际无毒类。经重组酵母处理的樱桃番茄果实,其在贮藏期间的品质指标与对照果实相比,没有明显的差异。更多还原

【Abstract】 Considerable amount of fruits and vegetables is lost due to decay after harvest. Biological control of postharvest decays (BCPD) with antagonistic yeasts have shown great potential as an alternative to synthetic fungicides for the control of postharvest decay of fruits and vegetables. However, the effectiveness of general yeast on postharvest decay control is not comparable to fungicides. The future expansion of it will largely depend on discovering new antagonists or improving its effectiveness. Using genetic manipulation to improving antagonists in BCPD is a new way in biological control of postharvest fruit. The current study was undertaken to investigate the possibility of enhance biocontrol activity of antagonists against fungal pathogens by expressing a DNA sequence in yeast to allow for the production of an antifungal peptide to produce an improved biocontrol. According to the experiment design, Cecropin A and PsdI gene was successfully expressed in methylotrophic yeast Pichia pastoris GS115. Recombination yeast GS115/CEC and GS115/PSD showed great biocontrol efficiency in inhibiting postharvest diseases of fruits and vegetables. The capability of growth of recombination yeast, the safety of yeasts, the efficacy of recombination yeasts alone or in combination with other control means for controlling postharvest disease of cherry tomatoes, oranges, pears and apples were tested. Meanwhile, the antibacterial mechanism of yeast and fermentation condition optimization of recombination yeasts were studied.The results were shown as below:1. We synthesized the gene fragment of peptides cecropin A and psdI encoding the mature peptide gene according to the genebank sequence of its, and then the fragments was connect to PGEM-T vector. Connect cecropin A and psdI gene to the expression vector of Pichia Pastoris after constructing on pPIC9K. After the linearization of it, punch with electricity law channel P. Pastoris GS115 into. Adopt G418 to gradient screen high transformation son of resisting etc. Through Triciene-SDS-PAGE, we confirmed the expression success of recombination yeasts after 72h induced by methanol. The experiment of fermentation and PCR method showed that recombination yeasts had a good stability.2. Improved expression level of recombinant yeasts was achieved by optimization both the growth phase and induction phase culture conditions. The results indicated that the optimum fermentation condition of the growth phase is as follows:2% glycerol,2% (NH4)2SO4, pH=6.5-7.5,30℃,72h. The test results of induction phase showed that the optimum desired fermentation condition is as follows:1% methanol,2%(NH4)2SO4, pH=7.0,30℃,72h. The fermented condition of recombinant yeasts after adopting the orthogonal experiment to optimize, The main factor that its influence ferments is as follows, temperature(30℃)> concentration of methanol (1%)> ferment time (4 d)> pH of the solution (5.5). Five times repeat fermentation experiments of strain GS115/CEC and GS115/PSD under the optimum conditions show that the average protein content is 15.1 and 11.3mg/L correspond with results of the orthogonal test.3. Recombination strain GS115/CEC and GS115/PSD showed great inhibition activity on four kinds of fungus(Alternaria alternata, Botrytis cinerea, Botrytis cinerea and Geotrichum citri-aurantii) in vitro tests. Recombination strains also showed a significant inhibitory effect against all four pathogens in fruits. In reducing the development of postharvest diseases of fruits and vegetables, including:black rot of cherry tomato, caused by A. alternata; gray mold decay of cherry tomato, pear and apple, caused by B. cinerea; green mold of apple and pear caused by P. expansum. The concentrations of yeasts had significant effects on biocontrol effectiveness to postharvest diseases of fruits:the higher the concentrations of recombination strains, the better the control efficacy.4. The results about the inhibition mechanism of the recombination yeasts showed that: besides of tranditional competing for nutrients and space, the results obtained by scanning electron microscopy and transmission electron microscopy showed that the morphology and inner structure of the fungi treated with recombination strain GS115/CEC changed obviously. Some of the cells were made abnormality, rupture or lysed.5. In an attempt to clarify the molecular mechanism of action, we examined the binding properties exerted by the peptide expressing by recombination yeast on DNA and RNA of fungal cells. Results showed the expression product can inhibit the migration of RNA. With the increasing of the quantity of peptides, the moving speed of nucleic acids in agarose became slower. Meantime, DNA and RNA of the fungal cells were degraded by the peptide. And then the metabolic process of fungi was destroyed and the fungi were killed. Moreover, the expression level of PR proteins that play an important role in the plant pathogen attack response was induced by recombination yeast. So, enhancing the resistance activity of fruits to pathogens may be another mechanism of control postharvest disease of fruits and vegetables of recombination strain.6. The efficacy of recombination yeasts on the control of postharvest decay of fruits was enhanced significantly by combining with metal salts (CaCl2), carbonate and ethanol in vivo test. The efficacy of GS 115/CEC for control of fruits postharvest disease was significantly improved when combined with these allogenic materials.7. On the safety test, the results of acute toxicity to mice (food standard) showed the dried powder of recombination yeasts were safe. The quality parameters of fruits treated with recombination yeasts were similar to those of control fruit, which suggested that the transformed yeast did not impair quality parameters of fruits.更多还原