【作者】 姜峰；

【导师】 郑晓冬； 陈集双；

【作者基本信息】 浙江大学 ， 食品科学， 2008， 博士

【摘要】 果实采后真菌性病害造成的经济损失是巨大的。长期以来防治果实的真菌病害一直是使用化学杀菌剂,而化学杀菌剂早就被证实有害于人体健康,因而开发安全无害的果蔬保鲜剂具有重要意义。利用具有拮抗活性的酵母菌种开展生物防治的方法是最有希望替代化学杀菌剂的方法之一。罗伦隐球酵母(Cryptococcus laurentii)是国内外研究较多的采后拮抗酵母。目前研究认为生防酵母预防采后病原菌的主要作用机制包括：营养与空间竞争、直接与病原微生物作用和诱导寄主抗性三个方面,其中营养与空间竞争被认为是生防酵母主要的作用方式。越来越多的证据表明,生防酵母可以诱导寄主产生抗性,并且这种抗性在采后储存中抵御采后病原微生物的侵染起到了重要的作用。本论文以番茄果实为实验材料,主要分为三部分。第一部分,以抑制差减杂交(SSH)和番茄基因芯片方法筛选出罗伦隐球酵母处理后樱桃番茄果实差异表达的基因；从芯片和SSH结果中选出4个差异表达的基因；第二部分,克隆了这四个基因的开放阅读框ORF (Open Reading Frame)并将其转入到拟南芥中以待对其功能进行深入研究。第三部分,用罗伦隐球酵母对番茄进行处理,检测樱桃番茄果实在罗伦隐球酵母和1-MCP单独和结合处理下对番茄果实抗性相关酶活性的影响；其主要研究结果如下：以生防菌处理后果实cDNA为’tester’,以无菌水处理的为‘driver’构建抑制差减杂交(SSH)文库。通过对SSH文库的筛选得到50个差异表达基因,其中33个为已知功能,在已知功能的基因中主要是抗性相关和信号转导相关的基因,而这些基因在其他的胁迫条件下也有上调表达,因此认为正是这些基因的上调表达,增强了番茄果实在生防菌处理后对采后病害的抗性。通过番茄基因芯片杂交,我们观察到在生防酵母处理后番茄果实约有4200个基因的表达发生了变化,约占番茄基因总数的12%。通过进一步的筛选芯片杂交结果,我们得到了531个差异表达明显的基因。其中上调表达基因219个,其中96为已知功能,123个功能未知；下调表达基因312个其中个168为已知功能,144个功能未知。在上调表达已知功能的基因中主要包括如下几类：基础代谢22个占已知基因10%；抗性相关基因17个占已知基因7.7%；信号转导10个占已知基因4.5%；；在下调表达已知功能的基因中主要包括如下几类：基础代谢41个占已知基因13%；光合作用24个占已知基因7.7%；能量代谢相关21个占已知基因6.7%。通过对基因芯片的结果分析,得到如下的结论：生防菌处理番茄果实后,可以激发SA介导的SAR反应,抑制JA/ET介导的其他抗性反应。而且生长素介导的抗性反应也可能在生防酵母处理下起到一定的作用。同时生防酵母处理后可以明显的抑制番茄果实中光合作用和能量代谢相关基因的表达。为进一步研究相关基因的功能,我们克隆得到了4个基因的ORF并构建了过表达载体,并将其转导拟南芥中,通过除草剂筛选,目前已经得到拟南芥转基因苗。以绿熟期樱桃番茄果实为试验材料,用1μl/L浓度1-甲基环丙烯(1-MCP)进行处理24小时,在室温条件下自然贮藏,研究1-MCP处理对番茄果实抗性相关酶：过氧化氢酶(CAT)、过氧化物酶(POD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)、超氧化物歧化酶(SOD)、脂肪氧合酶(LOX)活性和丙二醛(MDA)含量的影响。结果表明：1-MCP处理可以抑制LOX和PAL的活性；刺激CAT活性峰值提前；提高MDA含量；刺激PPO和POD活性上升,但是1-MCP处理对SOD活性影响不大。1-MCP和罗伦隐球酵母结合处理可以刺激组织中POD活性上升和MDA含量增加,抑制组织中SOD活性。而对其他酶的活性没有显著影响。更多还原

【Abstract】 Postharvest fungal diseases cause substantial economic losses world-widely. In view of the fungicide toxicity on the environment and human health, and the development of fungicide resistance by pathogens, great efforts have been made to exploit alternatives to the synthetic fungicides. The biological control by using antagonistic yeasts is one of the most promising means. Strains of Cryptococcus laurentii are well known postharvest biocontrol yeasts, which have been shown high antagonistic activity in various fruit. Antagonistic yeasts have been selected mainly for their activity does not generally depend on the production of toxic metabolites, which could have a negative environment or animal toxicological impact, and for their ability to colonize and grow rapidly in surface wounds and for there are generally poorly sensitive to fungicides. Different mechanisms of action have been suggested for antagonist yeasts, which could be complementary to each other. Competition for space and nutrients, which is believed to be the major mode of action of antagonistic yeasts. In addition, several studies have shown that the capability of producing and secreted cell wall-degrading enzymes may involved in the action of antagonistic yeasts. Also the induce resistance in the host tissue play important role in the action of antagonistic yeasts. The objective of this paper were:(1) using SSH methods and Tomato Genechip to isolated different expression genes from tomato fruit after treat by Cryptococcus laurentii, (2) select four differently expressed genes from results of SSH and Tomato Genechip, clone Open Reading Frame (ORF) and then transform to Arabidopsis, for further function analysis. (3) detect the activity changes of defense-related enzymes include CAT, SOD, PPO, PAL, POD, LOX and concentration changes of MDA after treated by Cryptococcus laurentii or 1-MCP alone and combine.SSH was performed with cDNA from cherry tomato fruit inoculated by water as the "driver" and cDNA from tomato fruit inoculated by Cryptococcus laurentii as the "tester". A selected 50 clones in the SSH library were sequenced. BLASTX results revealed that 33 cDNAs had significant sequence homologies with known sequences in the NCBI database. The identified cDNAs encoded proteins involved in cellular processes such as:primary metabolism; Ripen-related; signal transduction; Defense and pathogen-related or stress stimuli or wounding. Six clones were selected for temporal expression analysis using RT-PCR based on the results of the reverse Northern blot screens. Results from RT-PCR confirmed the differential expressions of these transcripts. The results show that a number of transcripts encoding proteins/enzymes which are known to be up-regulated under some biotic and abiotic stress are also up-regulated after the application of biological control yeast to cherry tomato fruit. The expression of these proteins might increase the fruit resistance towards postharvest pathogen infection and damage.To obtain an overall picture of gene regulation during biocontrol yeast treated, two independent microarray analyses were performed. To reduce experimental variation, two sets of twenty fruits were harvested from biocontrol yeast-treated and untreated(wounding control) fruit, respectively, at 24h post treatment. 531 genes were selected from Tomato Genechip, the number of genes with increased signals was 219,123 of 219 have unknown function and that with decreased signals was 312,144 of 312 have unknown function. The majority of the up-regulated genes seem to be related to three major biological changes induced by biocontrol yeast in the tomato fruit, primer metabolism (10%), defense-related (7.7%) and transcription (4.5%). The three largest functional categories (except for unclassified protein) of the down-regulated genes are primary metabolism (13%), photosynthesis (7.7%) and energy metabolism(6.7%). Results from Tomato Genechip indicated that Cryptococcus laurentii can induced SA mediate SAR reaction and suppressed JA/ET mediate resistance reaction, at same time suppressed genes expression involved in energy metabolism and photosynthesis.For further analysis selected genes functions ORF of four selected genes were cloned and transformed in to Arabidopsis. The transgenic plants have been obtained.In order to got insight view to the relative between 1-MCP treatment and fruit resistance to postharvest pathogens. Effects of 1-MCP on resistance-related enzymes include catalase(CAT), Peroxidase (POD), Polyphenoloxidase (PPO), Phenylalanineammonialyase (PAL), Superoxide dismutase (SOD, linoleate:oxygen oxidoreductase (LOX) and malondialdehyde (MDA) concentration of cherry tomato fruit were determined. 1-MCP treated can induce PPO and POD activity, increased the concentration of MDA, and suppressed LOX and PAL activity. 1-MCP treated have on effect on SOD activity in cherry tomato fruit. The peak of CAT activity was bring forward 12h ahead after treated by 1-MCP compare with the control.更多还原