苹果ASR基因家族的鉴定与表达分析
发布时间：2018-08-04 21:48
【摘要】：苹果(Malus domestica)是蔷薇科苹果属乔木,具有品种繁多、生态适应性强、营养价值高、耐储运性好和供应周期长等特点,世界上相当多的国家都将其列为主要消费品而大力发展。苹果斑点落叶病是一种世界性的真菌病害,在全球的苹果产区普遍发生,该病对苹果的质量和产量影响很大,严重时常造成早期落叶。因而,发掘抗性基因及胁迫相关基因开展抗病育种具有重要理论价值和实践意义。ASR(Abscisic acid、Stress and Ripening)基因是近几年来研究较多的一类基因,最早从番茄中发现,随后在其他物种中相继被发现。关于ASR基因的研究主要侧重于非生物胁迫,在生物胁迫方面的研究却相对较少,特别是关于苹果斑点落叶病的研究未见报道。本文从苹果斑点落叶病病原菌这类生物胁迫开始,研究这类基因,期望能从中找到一些参与此胁迫响应的主要基因从而继续探究此类基因在生物胁迫下的响应机制。本论文的主要研究内容包括以下三个方面:1、运用生物信息学的方法,从全基因组水平上鉴定苹果ASR基因家族成员,并对其系统发育树、理化性质、二级结构、基因结构、motif、亚细胞定位和蛋白质功能进行预测和分析;2、通过对'金冠'和'红星'两个品种的叶片离体接菌,利用体式显微镜观察并记录叶片上病斑的扩展情况和统计叶片的发病率;利用紫外分光光度计,对植物细胞内的5个防御酶POD、SOD、PAL、PPO和CAT活性进行测定;利用荧光定量qRT-PCR对苹果ASR基因家族成员在'金冠'和'红星,两个品种上受苹果斑点落叶病病原菌侵染后的表达模式进行了分析。主要结果如下:1.鉴定出了 5个ASR基因,并根据它们在染色体上的位置分别命名为ASR1、2、3、4和ASR5;苹果ASR基因家族为高度的亲水性蛋白,富含谷氨酸、组氨酸、甘氨酸、赖氨酸和丙氨酸;二级结构主要是α螺旋;亚细胞定位有3个基因定位于细胞核;预测的蛋白质功能中多数参与植物的生长过程,少数参与转录调控过程。从Ka/Ks上看,基因对ASR2/ASR3的值(2.07)大于另外三对基因的数值(0.67,0.47和0.71),说明ARS2和ASR3受到了正选择作用。从Ks来看,ASR2和ASR3值(0.05)小于另外两个值(0.74和1.51),很有可能是近期复制的;从系谱发生图上看,苹果家族成员都聚集在一起,表现出了物种特异性复制的特征;2.通过发病率和病情指数的统计,发现'金冠'是感病品种,'红星'是高感品种;通过病程的观察,发现'金冠'抗病菌的扩展阶段而'红星'抗病菌的侵染阶段;通过对生理指标的测定,发现5种防御酶的活性总体趋势是先升高后下降,金冠的抗病能力要比红星的强;利用qRT-PCR,计算出5个Md-ASR基因的相对表达量,结果表明ASR基因受到苹果斑点落叶病病原菌侵染后,基因具有不同的表达模式。在金冠中,ASR1在所有的时间点上都上调表达,而其它4个基因在时间点18 h和36 h都下调表达,然后在72 h时上调表达。在红星中,ASR1、2和3在所有的时间点都下调表达。ASR4和ASR5两个基因在36 h这个时间点上都上调表达,在18 h和72 h时都下调表达,而且它们的表达量变化相当低。这些基因表达量的变化说明了ASR基因有可能参与了植物应对病原菌胁迫的响应过程。
[Abstract]:Apple (Malus domestica) is a tree of the genus Rosaceae. It has many varieties, strong ecological adaptability, high nutritional value, good storage and transport resistance and long supply cycle. Many countries in the world have developed it as the main consumer goods. Apple spotted deciduous disease is a worldwide fungal disease and is produced in the world. It is common in the region that the disease has great influence on the quality and yield of apple, and it often causes early deciduous leaves. Therefore, it is of great theoretical and practical significance to explore resistance genes and stress related genes to carry out resistance breeding..ASR (Abscisic acid, Stress and Ripening) gene is one of the most studied genes in recent years, the earliest from tomato It was found in other species and was subsequently discovered in other species. The research on ASR gene mainly focused on abiotic stress, but the research on biological stress was relatively small, especially on the study of Apple spotted deciduous disease. This paper began to study these genes from the biological stress of the pathogen of Apple spotted deciduous disease. We can find some major genes involved in this stress response and continue to explore the response mechanism of this kind of gene under biological stress. The main contents of this paper include the following three aspects: 1, using bioinformatics to identify the members of the apple ASR gene family from the whole genome level and the phylogeny of its phylogeny tree. Properties, two level structure, gene structure, motif, subcellular localization and protein function, and 2, by observing and recording the spread of the leaf spots on the leaves and the incidence of the leaves by means of the body microscope, and using the ultraviolet spectrophotometer for the plant cell. 2 The activities of 5 defense enzymes, POD, SOD, PAL, PPO and CAT, were measured, and the expression patterns of apple ASR gene family members in 'golden crowns' and' red stars' and two varieties of Apple spotted deciduous pathogens were analyzed by fluorescence quantitative qRT-PCR. The main results were as follows: 1. identified 5 ASR genes and based on them on the chromosomes. The locations are named ASR1,2,3,4 and ASR5, respectively; the apple ASR gene family is highly hydrophilic protein, rich in glutamic acid, histidine, glycine, lysine and alanine; the two grade structure is mainly alpha helix; subcellular localization has 3 genes located in the nucleus; most of the predicted egg white matter is involved in plant growth and a small number of participants. Transcriptional regulation process. From Ka/Ks, the value of the gene to ASR2/ASR3 (2.07) is larger than the other three pairs of genes (0.67,0.47 and 0.71), indicating that ARS2 and ASR3 are positively selected. From Ks, the ASR2 and ASR3 values (0.05) are less than the other two values (0.74 and 1.51), and are likely to be replicated in the near future; from the genealogical map, the apple family members All gathered together to show the characteristics of species specific replication; 2. through the statistics of the incidence and disease index, it was found that the 'golden crown' was a susceptible variety, and the 'Red Star' was a highly susceptible variety; through the observation of the course of disease, the "golden crown" was found to be an extended stage of antigerms and the infection stage of the 'Red Star' resistant bacteria; by the measurement of physiological indexes, 5 kinds of prevention were found. The general trend of the activity of the Royal enzyme first increased and then declined, and the resistance of the gold crown was stronger than that of the red star. The relative expression of 5 Md-ASR genes was calculated using qRT-PCR. The results showed that the gene had different expression modes after the ASR gene was infected by the pathogen of apple spotted deciduous disease. In Jin Guanzhong, ASR1 was up-regulated at all time points. The expression, while the other 4 genes were downregulated at time points 18 h and 36 h, and then up-regulated at 72 h. In red stars, ASR1,2 and 3 down regulated the expression of.ASR4 and ASR5 two genes at the time point of 36 h, down the expression at 18 h and 72 h, and their expression changes were very low. The change of gene expression indicates that ASR gene may be involved in the response of plants to pathogen stress.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S661.1
本文编号：2165267
[Abstract]:Apple (Malus domestica) is a tree of the genus Rosaceae. It has many varieties, strong ecological adaptability, high nutritional value, good storage and transport resistance and long supply cycle. Many countries in the world have developed it as the main consumer goods. Apple spotted deciduous disease is a worldwide fungal disease and is produced in the world. It is common in the region that the disease has great influence on the quality and yield of apple, and it often causes early deciduous leaves. Therefore, it is of great theoretical and practical significance to explore resistance genes and stress related genes to carry out resistance breeding..ASR (Abscisic acid, Stress and Ripening) gene is one of the most studied genes in recent years, the earliest from tomato It was found in other species and was subsequently discovered in other species. The research on ASR gene mainly focused on abiotic stress, but the research on biological stress was relatively small, especially on the study of Apple spotted deciduous disease. This paper began to study these genes from the biological stress of the pathogen of Apple spotted deciduous disease. We can find some major genes involved in this stress response and continue to explore the response mechanism of this kind of gene under biological stress. The main contents of this paper include the following three aspects: 1, using bioinformatics to identify the members of the apple ASR gene family from the whole genome level and the phylogeny of its phylogeny tree. Properties, two level structure, gene structure, motif, subcellular localization and protein function, and 2, by observing and recording the spread of the leaf spots on the leaves and the incidence of the leaves by means of the body microscope, and using the ultraviolet spectrophotometer for the plant cell. 2 The activities of 5 defense enzymes, POD, SOD, PAL, PPO and CAT, were measured, and the expression patterns of apple ASR gene family members in 'golden crowns' and' red stars' and two varieties of Apple spotted deciduous pathogens were analyzed by fluorescence quantitative qRT-PCR. The main results were as follows: 1. identified 5 ASR genes and based on them on the chromosomes. The locations are named ASR1,2,3,4 and ASR5, respectively; the apple ASR gene family is highly hydrophilic protein, rich in glutamic acid, histidine, glycine, lysine and alanine; the two grade structure is mainly alpha helix; subcellular localization has 3 genes located in the nucleus; most of the predicted egg white matter is involved in plant growth and a small number of participants. Transcriptional regulation process. From Ka/Ks, the value of the gene to ASR2/ASR3 (2.07) is larger than the other three pairs of genes (0.67,0.47 and 0.71), indicating that ARS2 and ASR3 are positively selected. From Ks, the ASR2 and ASR3 values (0.05) are less than the other two values (0.74 and 1.51), and are likely to be replicated in the near future; from the genealogical map, the apple family members All gathered together to show the characteristics of species specific replication; 2. through the statistics of the incidence and disease index, it was found that the 'golden crown' was a susceptible variety, and the 'Red Star' was a highly susceptible variety; through the observation of the course of disease, the "golden crown" was found to be an extended stage of antigerms and the infection stage of the 'Red Star' resistant bacteria; by the measurement of physiological indexes, 5 kinds of prevention were found. The general trend of the activity of the Royal enzyme first increased and then declined, and the resistance of the gold crown was stronger than that of the red star. The relative expression of 5 Md-ASR genes was calculated using qRT-PCR. The results showed that the gene had different expression modes after the ASR gene was infected by the pathogen of apple spotted deciduous disease. In Jin Guanzhong, ASR1 was up-regulated at all time points. The expression, while the other 4 genes were downregulated at time points 18 h and 36 h, and then up-regulated at 72 h. In red stars, ASR1,2 and 3 down regulated the expression of.ASR4 and ASR5 two genes at the time point of 36 h, down the expression at 18 h and 72 h, and their expression changes were very low. The change of gene expression indicates that ASR gene may be involved in the response of plants to pathogen stress.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S661.1
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