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| 石果衣真菌膜联蛋白基因在酵母和拟南芥中表达初步研究 | |
| 其他题名 | A preliminary study on the expression of annexin gene from Endocarpon pusillum in yeast and arabidopsis |
| 张永利 | |
| 出版年 | 2017 |
| 学位类型 | 硕士 |
| 导师 | 魏江春 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 石果衣（Endocarpon pussilum）是一种生长在荒漠地区的地衣，它是由其共生菌（Endocarpon pussilum）和共生藻——柯氏复球藻（Diplosphaeria chodatii）组成的一种稳定的共生生态系统。之前的研究发现石果衣共生菌具有极强的抗旱能力，随着石果衣共生菌基因组和转录组的完成，可以在分子水平上对石果衣抗逆机制进行研究。本文选取了9个PEG胁迫条件下，转录组中上调表达的基因进行研究。由于石果衣目前缺乏有效的遗传操作方法，我们在酿酒酵母（Saccharomyces cerevisiae）中进行异源表达分析。结果发现：过表达Epnhx、Epmap和Epann 3个基因能分别显著提高酿酒酵母对氯化钠、重金属和高温的抵抗能力，其中以Epann抗逆能力最为突出。\n 对Epann基因进一步研究后发现：过氧化氢、氯化钠和PEG胁迫都能够诱导石果衣共生菌内Epann基因的上调表达。Epann基因的编码产物是一种膜联蛋白EpANN，正常生长条件下，EpANN定位于细胞质，但在高温胁迫条件下，EpANN会发生明显的聚集。异源表达Epann能够提高酵母对高温、过氧化氢和氯化钠的抵抗能力；同时能降低酿酒酵母在高温胁迫条件下细胞内的活性氧（ROS）含量，提高超氧化物歧化酶（SOD）和过氧化氢酶（CAT）的活性。对Epann抗逆机制进行研究后发现：EpANN具有过氧化物酶活性，能够帮助清除细胞内的ROS，降低细胞在胁迫条件下的氧化损伤。并且，我们的研究首次在真菌内发现膜联蛋白具有分子伴侣活性，能够提高真菌对高温胁迫的抵抗能力。同时证明EpANN具有钙离子依赖的磷脂结合特性，并且发现过表达Epann能够提高其他抗逆基因在胁迫条件下的表达水平。\n 此外，在拟南芥中表达Epann后，拟南芥在过氧化氢胁迫条件下的发芽率和长势都优于野生型，对干旱胁迫也更加耐受。 |
| 英文摘要 | The lichen-forming fungus Endocarpon pusillum (Verrucariales, Ascomycota) was isolated from the desert dominant species, living with the green alga Diplosphaera chodatii to form a stable symbiotic eco-system. Previous physiological investigations indicated that this fungus is extremely drought-tolerant. The availability of its genome and transcriptome information has made it an attractive model for studying the abiotic stress tolerance mechanisms in lichenized fungi and lichen. Due to its slow growth rate and the lack of genetic transformation system, functional analysis of the candidate stress-related genes of E. pusillum relies on the heterologous expression system of Saccharomyces cerevisiae. Nine differentially expressed genes from the PEG-treatment transcriptome were chosen and investigated in this study. The results showed that overexpression of Epnhx, Epmap and Epann could improve the resistance of S. cerevisiae to sodium chloride, heavy metal and high temperature stress respectively, among which the resistance ability of Epann was the most prominent and the functions of Epann were further studied.The results of Epann expression pattern assay indicated that hydrogen peroxide, sodium chloride and PEG could induce the up-regulation of its expression in Endocarpon pusillum. The protein product of Epann is an annexin, EpANN locates in the cytoplasm under normal growth condition. However, a significant aggregation of EpANN was observed under heat stress condition in S. cerevisiae cells. Overexpression of Epann enhanced the tolerance of S. cerevisiae to high temperature, hydrogen peroxide and sodium chloride stresses. At the same time, it reduced the reactive oxygen species (ROS) content and increased the activity of SOD and CAT in S.cerevisiae. The peroxidase activity of EpANN was found, which could repress the accumulation of ROS and reduce the oxidative damage to cells. Significantly, we report here for the first time the chaperone activity of fungal annexins and show that this activity maybe one of the primary mechanisms of EpANN in resistant to abiotic stresses. We also proved that EpANN could bind to phospholipid in a calcium-dependent manner, and transgenic yeast had a higher expression level of stress-resistant genes than empty vector control under heat stress. In addition, overexpression of Epann improved the resistance of Arabidopsis thaliana to hydrogen peroxide, and transgenic A. thaliana were more drought tolerant than wild-type plants. |
| 中文关键词 | 石果衣 ; 抗逆 ; 膜联蛋白 ; 转基因酵母 |
| 英文关键词 | Endocarpon pusillum stress resistance annexin，transgenic yeast |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 微生物学 |
| 来源机构 | 中国科学院微生物研究所 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/287924 |
| 推荐引用方式 GB/T 7714 | 张永利. 石果衣真菌膜联蛋白基因在酵母和拟南芥中表达初步研究[D]. 中国科学院大学,2017. |
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