利用基因芯片解析金柑和纽荷尔应答溃疡病的转录差异

Analysis of Different Transcriptional Responses to Citrus Canker between Meiwa and Newhall Via Microarray

作者: 专业:果树学 导师:刘继红 年度:2010 学位:硕士  院校: 华中农业大学

Keywords

Citras canker, differentially expressed genes, Microarray, Bioinformatics

        柑橘溃疡病是柑橘的主要病害之一,本研究以柑橘溃疡病感病品种纽荷尔甜橙和抗性品种宁波金柑为材料,使用基因芯片技术分析两者在接种溃疡病致病菌后的基因表达谱差异,并使用半定量PCR验证了基因芯片结果的可靠性,最后应用生物信息学对差异表达基因进行功能分析。结果如下:1.柑橘溃疡病致病菌离体接种试验表明,接种后叶片出现典型病发症状的时间在3-5天,第5天发病率为近100%。2.芯片结果利用R软件bioconductor平台下的RankProd芯片统计软件包对Affymetrix芯片杂交结果的CEL文件进行自主分析,以为标准筛选差异基因,筛选结果表明:纽荷尔上调基因610个,下调基因714个;金柑上调基因530个,下调基因264个;其中金柑与纽荷儿共上调基因150个,共下调基因80个;金柑中上调倍数高于纽荷尔的基因97个,金柑中上调纽荷尔中下调的基因为5个;金柑中特异上调基因376个。取统计分析结果中二者共上调且金柑中上调倍数高于纽荷尔的基因97个,金柑上调纽荷尔下调的基因5个,金柑中特异上调的基因376个,共478个基因进行生物信息学分析,用Annot8r生物信息学软件对478个基因进行了功能注释包括GO、EC。结果表明其中存在许多已经报道过具有抗病相关功能的基因,如水杨酸、茉莉酸、类黄酮、HR、SAR、气孔调节、激素运输、乙烯、类苯基丙烷、木质素等相关抗病基因。除此之外,在研究中还有许多未报道的但差异表达显著的基因,其功能有待于深入研究。3.选取了17个基因进行Semi-Quantitative RT-PCR验证芯片可靠性,结果表明17个基因Semi-Quantitative RT-PCR结果表明同芯片结果,证明了芯片结果可靠性。
    Citrus canker is one of the main diseases affecting citrus production. In this study, microarray was employed to analyze the differential gene expression patterns of two varieties, Newhall (canker-tolerant) and Kumquat (canker-sensitive), after being inoculated with the canker bacteria. Subsequently, bioinformatics and semi-quantitive RT-PCR were conducted to analyze the results and verify the differentially expressed genes, respectively. The main results are as follows:1. In vitro inoculation test showed that after inoculation of the citrus canker pathogen, typical symptoms appeared within 3-5 days, and the incidence rate was nearly 100% at the fifth day.2. The CEL files of the Affymetrix chips were analyzed independently by RankProd statistical package downloaded from R&bioconductor software platform. Differentially expressed genes were screened under the standard of Fold-Change|>=4 and FDR<=0.5. The results showed that:there are 610 up-regulated and 714 down-regulated genes in Newhall, while 530 and 264 in kumquat respectively. Together, there are 150 up-regulated and 80 down-regulated genes in both varieties. Out of the 150,97 up-regulated genes expression are higher in Kumquat. Five genes are up-regulated in Kumquat but down-regulate in Newhall, and 376 up-regulated genes in Kumquat have no match in Newhall. Taken the 97,5,376 together, these 478 genes were selected for further bioinformatics analysis, such as functional annotation by GO, EC, with Annot8r. The results showed that many of the genes, as reported before, were associated with disease resistance, such as salicylic acid, jasmine acid, flavonoids, HR, SAR, stomatal regulation, hormones transport, ethylene, class-propane, and lignin. In addition, in our study, there are many differently expressed genes whose functions may have roles in Citrus canker resistance, needing to be further researched.3. Seventeen genes were selected for semi-Quantitative RT-PCR, whose expressions were consistent with the results of gene chip, indicating a certain reliability of microarray.
        

利用基因芯片解析金柑和纽荷尔应答溃疡病的转录差异

摘要6-7
Abstract7
缩略词表8-9
1 前言9-15
    1.1 课题的提出9-10
    1.2 柑橘溃疡病国内外研究进展10-11
        1.2.1 柑橘溃疡病的发生与危害10
        1.2.2 柑橘溃疡病的防治10-11
    1.3 基因芯片技术11-12
    1.4 基因芯片技术的特点12
    1.5 基因芯片技术在植物抗病研究中的应用12-13
    1.6 生物信息学在基因芯片中的应用13-14
    1.7 本研究的目的和意义14-15
2 材料与方法15-24
    2.1 试验材料15
        2.1.1 叶片与柑橘溃疡致病菌15
        2.1.2 主要软件/操作平台15
    2.2 试验方法15-24
        2.2.1 柑橘溃疡病致病菌的纯化保存15
        2.2.2 叶片离体接种15-16
        2.2.3 接种后叶片的柑橘溃疡病致病菌鉴定16-18
            2.2.3.1 材料16-18
        2.2.4 金柑与纽荷尔表达谱芯片杂交分析18-19
        2.2.5 Semi-Quantitative RT-PCR验证芯片可靠性19-24
3 结果与分析24-36
    3.1 柑橘溃疡病致病菌纯化鉴定24
    3.2 金柑和纽荷尔叶片离体接种发病情况24
    3.3 柑橘溃疡病致病菌阳性检测24-25
    3.4 表达谱芯片杂交分析结果25-36
        3.4.1 芯片原始扫描图像25
        3.4.2 芯片信号散点图(Scatter Graph)25-26
        3.4.3 差异基因的筛选26
        3.4.4 478个差异基因聚类分析26-27
        3.4.5 差异表达基因GO分类与分析27
        3.4.6 抗病相关酶类分析27-32
        3.4.7 表达谱芯片结果可靠性验证32-36
4 讨论36-42
    4.1 溃疡病刺激诱导的SAR、水杨酸、茉莉酸等相关基因36-37
    4.2 溃疡病诱导的调节生物学过程的基因37-38
    4.3 细胞分化相关基因38-39
    4.4 细胞组成氨基酸代谢和次生代谢相关基因39-40
    4.5 酶相关的差异表达基因40-42
5 结论42-43
参考文献43-48
图版和图版说明48-51
致谢51-52
附录52-62
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