【摘要】：蘋果(Malus domestica)是薔薇科蘋果屬喬木,具有品種繁多、生態(tài)適應(yīng)性強(qiáng)、營(yíng)養(yǎng)價(jià)值高、耐儲(chǔ)運(yùn)性好和供應(yīng)周期長(zhǎng)等特點(diǎn),世界上相當(dāng)多的國(guó)家都將其列為主要消費(fèi)品而大力發(fā)展。蘋果斑點(diǎn)落葉病是一種世界性的真菌病害,在全球的蘋果產(chǎn)區(qū)普遍發(fā)生,該病對(duì)蘋果的質(zhì)量和產(chǎn)量影響很大,嚴(yán)重時(shí)常造成早期落葉。因而,發(fā)掘抗性基因及脅迫相關(guān)基因開展抗病育種具有重要理論價(jià)值和實(shí)踐意義。ASR(Abscisic acid、Stress and Ripening)基因是近幾年來(lái)研究較多的一類基因,最早從番茄中發(fā)現(xiàn),隨后在其他物種中相繼被發(fā)現(xiàn)。關(guān)于ASR基因的研究主要側(cè)重于非生物脅迫,在生物脅迫方面的研究卻相對(duì)較少,特別是關(guān)于蘋果斑點(diǎn)落葉病的研究未見報(bào)道。本文從蘋果斑點(diǎn)落葉病病原菌這類生物脅迫開始,研究這類基因,期望能從中找到一些參與此脅迫響應(yīng)的主要基因從而繼續(xù)探究此類基因在生物脅迫下的響應(yīng)機(jī)制。本論文的主要研究?jī)?nèi)容包括以下三個(gè)方面:1、運(yùn)用生物信息學(xué)的方法,從全基因組水平上鑒定蘋果ASR基因家族成員,并對(duì)其系統(tǒng)發(fā)育樹、理化性質(zhì)、二級(jí)結(jié)構(gòu)、基因結(jié)構(gòu)、motif、亞細(xì)胞定位和蛋白質(zhì)功能進(jìn)行預(yù)測(cè)和分析;2、通過(guò)對(duì)'金冠'和'紅星'兩個(gè)品種的葉片離體接菌,利用體式顯微鏡觀察并記錄葉片上病斑的擴(kuò)展情況和統(tǒng)計(jì)葉片的發(fā)病率;利用紫外分光光度計(jì),對(duì)植物細(xì)胞內(nèi)的5個(gè)防御酶POD、SOD、PAL、PPO和CAT活性進(jìn)行測(cè)定;利用熒光定量qRT-PCR對(duì)蘋果ASR基因家族成員在'金冠'和'紅星,兩個(gè)品種上受蘋果斑點(diǎn)落葉病病原菌侵染后的表達(dá)模式進(jìn)行了分析。主要結(jié)果如下:1.鑒定出了 5個(gè)ASR基因,并根據(jù)它們?cè)谌旧w上的位置分別命名為ASR1、2、3、4和ASR5;蘋果ASR基因家族為高度的親水性蛋白,富含谷氨酸、組氨酸、甘氨酸、賴氨酸和丙氨酸;二級(jí)結(jié)構(gòu)主要是α螺旋;亞細(xì)胞定位有3個(gè)基因定位于細(xì)胞核;預(yù)測(cè)的蛋白質(zhì)功能中多數(shù)參與植物的生長(zhǎng)過(guò)程,少數(shù)參與轉(zhuǎn)錄調(diào)控過(guò)程。從Ka/Ks上看,基因?qū)SR2/ASR3的值(2.07)大于另外三對(duì)基因的數(shù)值(0.67,0.47和0.71),說(shuō)明ARS2和ASR3受到了正選擇作用。從Ks來(lái)看,ASR2和ASR3值(0.05)小于另外兩個(gè)值(0.74和1.51),很有可能是近期復(fù)制的;從系譜發(fā)生圖上看,蘋果家族成員都聚集在一起,表現(xiàn)出了物種特異性復(fù)制的特征;2.通過(guò)發(fā)病率和病情指數(shù)的統(tǒng)計(jì),發(fā)現(xiàn)'金冠'是感病品種,'紅星'是高感品種;通過(guò)病程的觀察,發(fā)現(xiàn)'金冠'抗病菌的擴(kuò)展階段而'紅星'抗病菌的侵染階段;通過(guò)對(duì)生理指標(biāo)的測(cè)定,發(fā)現(xiàn)5種防御酶的活性總體趨勢(shì)是先升高后下降,金冠的抗病能力要比紅星的強(qiáng);利用qRT-PCR,計(jì)算出5個(gè)Md-ASR基因的相對(duì)表達(dá)量,結(jié)果表明ASR基因受到蘋果斑點(diǎn)落葉病病原菌侵染后,基因具有不同的表達(dá)模式。在金冠中,ASR1在所有的時(shí)間點(diǎn)上都上調(diào)表達(dá),而其它4個(gè)基因在時(shí)間點(diǎn)18 h和36 h都下調(diào)表達(dá),然后在72 h時(shí)上調(diào)表達(dá)。在紅星中,ASR1、2和3在所有的時(shí)間點(diǎn)都下調(diào)表達(dá)。ASR4和ASR5兩個(gè)基因在36 h這個(gè)時(shí)間點(diǎn)上都上調(diào)表達(dá),在18 h和72 h時(shí)都下調(diào)表達(dá),而且它們的表達(dá)量變化相當(dāng)?shù)�。這些基因表達(dá)量的變化說(shuō)明了ASR基因有可能參與了植物應(yīng)對(duì)病原菌脅迫的響應(yīng)過(guò)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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S661.1

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