甘薯徐薯18和徐781淀粉合成相关差异表达基因分析及IbFBA基因克隆

发布时间：2018-01-27 23:44

本文关键词： 甘薯基因差异表达淀粉合成果糖-1 6-二磷酸醛缩酶逆境胁迫　出处：《中国农业科学院》2016年硕士论文　论文类型：学位论文

【摘要】：转录组测序技术是获取功能基因简单快捷的方法,并可根据转录组测序结果估算出基因的差异表达水平。本实验室对淀粉含量中等的徐薯18和高淀粉含量的徐781进行转录组测序,从中获得14个与淀粉合成相关的差异表达片段。为进一步了解这些基因的功能特性,本研究采用qRT-PCR方法研究这些基因在甘薯膨大各时期的表达模式,分析基因表达量与淀粉含量的内在联系,并对IbFBA2和IbFBA5两个基因进行克隆与初步功能验证。主要结果如下:1.根据qRT-PCR结果,这14个基因共呈现出6种不同的表达模式,即表达量逐渐上升、表达量不断下降、先上升-再下降、先下降-再上升、上升-下降-上升和下降-上升-下降。同一基因在不同品种之间、以及同一品种不同组织之间呈现不同的表达模式。其中NMT3在两个品种的两个部位间表现出4种不同的表达模式,其他基因均呈现出2到3种不同的表达模式。2.徐薯18叶片中的淀粉含量变化趋势与其叶片中IbFBA2的表达模式相同,皆为先下降后上升的模式;徐薯18薯块中淀粉含量的变化趋势与其薯块中GLDP1、IbAGPb1A、SBPas、T1-30106和T2-33205的表达模式相同,皆为逐渐增加。徐781叶片中淀粉含量变化与其叶片中IbFBA2、NMT3、SBPase、XYL1、T1-23698和T2-33205的表达模式相同,皆为先下降后上升;徐781薯块中淀粉含量的变化趋势与其薯块中IbAGPb1A、IbFBA5、MAP65-1、NMT2、T1-23698和T2-33205的表达模式相同,皆为先上升后下降。综合两个品种来看,IbFBA2在两个品种叶片中表达模式均与淀粉变化情况吻合;IbAGPb1A和T2-33205在两个品种薯块中表达模式均与淀粉变化情况吻合。另外,XYL1在不同时期的表达水平与蔗糖积累的变化趋势一致。3.从徐781中克隆到IbFBA2和IbFBA5。IbFBA2的cDNA全长1617 bp,开放阅读框为1197bp,编码398个氨基酸残基,含5个外显子和4个内含子,预测其亚细胞定位到质体中;IbFBA5的cDNA全长1341 bp,开放阅读框1074 bp,编码357个氨基酸残基,含3个外显子和2个内含子,预测其亚细胞定位到胞质中。4.分析IbFBA2和IbFBA5在栽后70天徐781的7个部位的表达情况发现,两个基因均具有组织表达特异性,IbFBA2主要在地上部表达量高,块根中表达量低;IbFBA5在叶片、储藏根、牛蒡根中表达量高,在花中表达量最低。5.IbFBA2和IbFBA5均能响应ABA处理以及盐、模拟干旱胁迫,但两基因响应胁迫的表达模式不同,IbFBA2的表达量皆呈先下降再上升的模式;而IbFBA5的表达量则显著高于0 h的表达量。6.构建过表达载体pGWB12::IbFBA2和pGWB12::IbFBA5,并瞬时转化烟草叶片,由碘染结果推测IbFBA可能不是淀粉合成的主效基因;转基因拟南芥的互补实验表明,IbFBA5响应ABA处理,并使转基因株系的根长恢复到野生型的表型。
[Abstract]:Transcriptome sequencing is a simple and rapid method for obtaining functional genes. The differentially expressed level of the gene could be estimated according to the results of transcriptome sequencing. Xushu 18 with medium starch content and Xu 781 with high starch content were sequenced in our laboratory. Fourteen differentially expressed fragments related to starch synthesis were obtained to further understand the functional characteristics of these genes. In this study, qRT-PCR method was used to study the expression patterns of these genes in different stages of sweet potato expansion, and to analyze the relationship between the amount of gene expression and the content of starch. The main results are as follows: 1. According to the results of qRT-PCR. These 14 genes showed six different expression patterns, that is, the expression level gradually increased, the expression level decreased, first ascended-then decreased, first-then increased-then increased. Rise-down-up-and-down-down-down-down-down. the same gene is between different varieties. The expression patterns of NMT3 were different among different tissues of the same variety, and there were 4 different expression patterns between the two parts of the two cultivars. All the other genes showed 2 to 3 different expression patterns. 2. The change trend of starch content in Xushu 18 leaves was the same as the IbFBA2 expression pattern in its leaves. The change trend of starch content in Xushu 18 potato was the same as the expression pattern of IbAGPb1 / IbAGPb1 / SB Pasa T1-30106 and T2-33205 in tuber. The changes of starch content in X781 leaves and IbFBA2NMT3NMT3NMT3 SBPasePaseXYL1 in X781 leaves were all increased gradually. The expression patterns of T1-23698 and T2-33205 were the same. The change trend of starch content in Xu781 tuber and IbAGPb1An IbFBA5 MAP65-1 NMT2 in tuber. The expression patterns of T1-23698 and T2-33205 were the same. The expression patterns of IbFBA2 in the leaves of the two varieties were consistent with the changes of starch. The expression patterns of IbAGPb1A and T2-33205 in the two varieties were consistent with the changes of starch. The expression level of XYL1 at different stages was consistent with the change trend of sucrose accumulation. The total length of cDNA 1617 was cloned from Xu 781 and cloned into IbFBA2 and IbFBA5.IbFBA2. Bp. The open reading frame was 1197bp, encoding 398 amino acid residues, including 5 exons and 4 introns, which were predicted to be located in the plastids. The cDNA of IbFBA5 is 1341 BP, open reading frame 1074 BP, encoding 357 amino acid residues, including 3 exons and 2 introns. The expression of IbFBA2 and IbFBA5 in 7 parts of Xu781 were analyzed 70 days after transplanting. The results showed that the two genes were tissue-specific. The expression of IbFBA2 was high in shoot and low in root. IbFBA5 expression was high in leaves, storage roots and burdock roots, and the lowest in flowers. IbFBA2 and IbFBA5 could respond to ABA treatment and salt, simulating drought stress. However, the expression patterns of IbFBA2 were different in response to stress, and the expression level of IbFBA2 decreased first and then increased. The expression of IbFBA5 was significantly higher than that of 0 h. 6. The overexpression vectors pGWB12::IbFBA2 and pGWB12::IbFBA5 were constructed. The results of iodine staining suggested that IbFBA might not be the main gene of starch synthesis. The complementary experiment of transgenic Arabidopsis thaliana showed that IbFBA5 responded to ABA treatment and restored the root length of transgenic lines to the wild-type phenotype.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S531;Q943.2

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