轮纹病致病菌Botryosphaeria dothidea侵染对苹果抗性反应、乙烯合成和相关基因表达的影响

发布时间：2018-08-29 10:09

【摘要】：苹果轮纹病是我国苹果产区的主要病害,属于真菌性病害,主要危害枝干和果实,为果品生产造成重大威胁。因此研究苹果果实与轮纹病菌之间的关系具有重要意义。我们通过研究和解析苹果抗病及免疫机理,分析苹果在受到病菌侵染后的各种生理活动和胁迫应答来挖掘苹果抗病基因和了解苹果抗病机制。苹果抗病基因的挖掘是进行品种改良的基础,对于培育抗病品种以及高效生产优质苹果具有重要的意义,同时该研究也对深入了解苹果的抗病基因具有重要的意义。为苹果优新抗病种质资源的培育提供科技支撑和依据,服务我国苹果产业发展。本实验以‘富士’和‘金冠’两种质地不同的苹果果实为材料,通过对苹果接种轮纹病菌后,测定果实乙烯释放量、硬度、基因相对表达量来进行苹果抗病性的系统研究,并对乙烯合成相关基因、细胞壁软化基因和PR家族基因进行深入研究。结果表明相关基因被病菌大量诱导表达,两个品种在抗苹果轮纹病方面存在显著差异,且苹果在不同发育时期的抗病反应非常复杂。针对病程相关基因在苹果抗病中的表达,我们从苹果中筛选出MdPR5和MdPR8,通过植物RNA的提取、PCR技术和基因克隆等方法,获得苹果中基因的cDNA序列,并对之进行了序列对比分析、基因表达和转基因分析,通过分子生物学手段验证不同种类苹果中抗病基因的表达。主要研究结果如下:1.植物体在逆境胁迫下的活性氧积累情况。以‘嘎拉’组培苗为试材,对叶片和植物接种轮纹病菌和flg22处理后,用DAB染色法检测活性氧的产生情况。结果叶片在受到轮纹病菌和flg22处理后有明显的黄色沉淀颗粒,黄色颗粒越多说明活性氧产生的越多,植株在受到flg22处理后茎部发红,说明叶片和植株都发生了逆境响应。2.轮纹病菌接种富士和金冠两个品种的苹果果实,在幼果期、膨大期、成熟期三个发育时期的乙烯释放速率、硬度变化、病斑大小差异明显,且用1-MCP处理接菌的果实,抑制乙烯释放的效果在不同品种的不同时期也不同,以金冠成熟期的变化最显著,接菌后金冠果实的病斑和硬度都明显小于富士的。对乙烯合成相关基因MdERF2、MdACS4、MdCTR1、MdACS5a、MdACS5b进行qRT-PCR分析,结果表明在成熟期,MdACS5a、MdACS5b这两个基因在轮纹病菌诱导下表达量明显升高。研究1-MCP对基因表达量的影响,发现1-MCP对MdACS5a、MdACS5b表达量的升高有显著抑制作用,1-MCP对MdACS4表达的抑制作用作用在金冠成熟期第六天较明显。3.接菌的富士和金冠两种苹果果实,在不同发育时期对细胞壁软化基因AFS1、PG1和PRs中的MdPR5、MdPR8、MdPR4进行qRT-PCR分析。发现接菌后幼果期和膨大期细胞壁软化基因相对表达量有明显升高。PRs相关基因的表达量在病菌侵染下也明显增高,这表明PRs具有抗病性。4.克隆MdPR5和MdPR8两个病程相关基因,进行序列分析和表达分析,并进行原核诱导和SDS-PAGE分析。为苹果抗病基因的挖掘和苹果品种改良做出基础性工作。并通过氨基酸序列对比分析发现MdPR5的氨基酸序列与苹果、拟南芥、梨、i卙r的某些序列相似性很高。
[Abstract]:Apple rot is a major disease of apple in China. It is a fungal disease which mainly damages branches and fruits and poses a great threat to fruit production. The subsequent physiological activities and stress responses were used to discover Apple resistance genes and understand the mechanism of Apple disease resistance. Significance. To provide scientific and technological support and basis for the cultivation of new apple germplasm resources, and serve the development of apple industry in China. The results showed that the genes related to ethylene biosynthesis, cell wall softening and PR family genes were induced and expressed in large quantities by pathogens. There were significant differences in resistance to apple ring rot between the two cultivars, and the resistance responses of apples at different developmental stages were very complex. We screened out MdPR5 and MdPR8 from apple, obtained the gene cDNA sequence of Apple by the methods of plant RNA extraction, PCR and gene cloning, and carried on the sequence comparison analysis, gene expression and transgenic analysis, and verified the resistance of different apple species through molecular biological means. The main results are as follows: 1. Accumulation of reactive oxygen species (ROS) in plants under stress. The leaves and plants were inoculated with Rhizoctonia solani and flg22, and the production of ROS was detected by DAB staining. The more precipitated particles and yellow particles, the more reactive oxygen species produced, and the redder the stem was treated with flg22, indicating that both leaves and plants had undergone stress response. 2. The ethylene release rate and hardness of apple fruits inoculated with Fuji and Golden Crown were changed during the three development stages of young fruit, expansion and maturity. The effect of 1-MCP on inhibiting ethylene release was also different in different cultivars at different stages. The changes in the ripening stage of golden crown were the most significant. The plaque and hardness of golden crown fruit after inoculation were significantly less than Fuji. For the genes related to ethylene synthesis, MdERF2, MdACS4, MdCTR1, MdACS5a and MdACS5b, the qRT-ACS5b was used. PCR analysis showed that the expression of MdACS5a and MdACS5b increased significantly in the mature stage. The effect of 1-MCP on the expression of MdACS5a and MdACS5b was studied. It was found that 1-MCP significantly inhibited the expression of MdACS5a and MdACS5b, and 1-MCP significantly inhibited the expression of MdACS4 on the sixth day of golden crown. The relative expression of cell wall softening genes AFS1, PG1 and PRs, MdPR5, MdPR8, and MdPR4 in Fuji and Golden Crown apple fruits inoculated with bacteria were analyzed by qRT-PCR at different developmental stages. The results showed that PRs had disease resistance. 4. Cloning MdPR5 and MdPR8 genes, sequence analysis and expression analysis, prokaryotic induction and SDS-PAGE analysis were carried out. The basic work was done for the excavation of disease resistance genes and the improvement of apple varieties. The amino acid sequences of MdPR5 were compared with those of apple and Yunnan by amino acid sequence analysis. The sequence similarity of mustard, pear, I R is very high.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S436.611

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