拟南芥中LjBIO同源基因AtKIX8和AtKIX9启动子的克隆及表达分析
发布时间：2018-07-17 01:19
【摘要】：豆科植物百脉根的BIO ORGANS基因(LjBIO)已经被证实与花器官对称性和大小调控有关,目前有关于LjBIO基因具体功能和调控机制的研究报告很少。拟南芥AtKIX8和AtKIX9基因与百脉根LjBIO基因属同源基因,编码产物属于KIX蛋白家族成员,被预测参与了植物器官大小及形态调控,但该蛋白家族的生物学功能及其作用机理尚不清楚。调控植物的器官大小在生产实践中有广泛的应用前景,对研究植物形态外观、生存繁殖、胁迫抗性等品质的遗传有重要指导意义。启动子是调控基因表达的重要顺式元件,决定着特定基因的定时、定位、定量表达,对植物启动子的研究有助于了解该基因的转录调控机制及其表达模式,并为研究基因的功能提供线索。本研究以模式植物拟南芥为研究材料,对LjBIO同源基因AtKIX8和AtKIX9的启动子进行了克隆和表达分析研究,期望揭示该类基因调节器官大小的作用机制,并为进一步阐明LjBIO基因的功能及机理提供更多的线索。现阶段取得的主要研究成果如下：1、利用PlantCARE在线预测软件对AtKIX8和AtKIX9启动子序列进行转录调控元件分析。分析结果表明,它们的启动子序列中含有生长素(IAA)响应元件、脱落酸(ABA)响应元件、赤霉素(GA)响应元件、低温胁迫响应元件等元件。2、克隆了拟南芥AtKIX8和AtKIX9基因启动子各长、短两个片段,分别命名为pKIX8L、pKIX8S、pKIX9L和KIX9S,大小分别为2851 bp、954 bp、2982bp和1379 bp。并构建相应接GUS报告基因的表达载体pKIX8:GUS、 PKIX8S:GUS、pKIX9L:GUS、pKIX9S:GUS,成功将这4个表达载体导入根癌农杆菌GV3101。3、通过花序侵染法将pKIX8L:GUS、pKIX8S:GUS、pKIX9L:GUS和pKIX9S:GUS这4个表达载体遗传转化拟南芥,用Hyg筛选转基因阳性植株,最终获得7株转pKIX8L:GUS的纯合转基因植株,6株转pKIX8S:GUS的纯合转基因植株,6株转pK1X9L:GUS的纯合转基因植株,2株转pK1X9S:GUS的纯合转基因植株。4、GUS染色结果显示,在AtKIX8基因启动子的驱动下,GUS基因主要集中在10天幼苗的茎尖分生组织区、下胚轴中柱以及幼叶的主脉等维管系统中,以及4周开花植物的叶片主脉和茎中有强表达。而在AtK1X9基因启动子驱动下,未检测到GUS基因在任何部位有表达。5、选取GUS染色阳性的转基因纯合体进行实验,定量结果显示在生长素(IAA 10μmol/L)和低温(4℃)处理后GUS基因的表达量高,而在脱落酸(ABA 200μmol/L)和赤霉素(GA 50μmol/L)处理后GUS基因表达量并无显著差异。
[Abstract]:The Bio ORGANS gene (LjBIO) of Leguminosae has been proved to be related to floral organ symmetry and size regulation. There are few studies on the specific function and regulation mechanism of LjBIO gene. Arabidopsis thaliana AtKIX8 and AtKIX9 genes belong to the homologous genes of LjBIO gene and belong to the KIX protein family, which are predicted to participate in the regulation of plant organ size and morphology. However, the biological function and the mechanism of the protein family are not clear. The regulation of plant organ size has a broad application prospect in production practice, which has an important guiding significance in studying the inheritance of plant morphological appearance, survival and reproduction, stress resistance and so on. Promoter is an important cis-element to regulate gene expression, which determines the timing, location and quantitative expression of specific gene. The study of plant promoter is helpful to understand the transcriptional regulation mechanism and expression pattern of the gene. It also provides clues for the study of gene function. In this study, the promoter of LjBIO homologous genes AtKIX8 and AtKIX9 were cloned and expressed in Arabidopsis thaliana, in order to reveal the mechanism of regulating organ size. It provides more clues for further elucidating the function and mechanism of LjBIO gene. The main research results obtained at present are as follows: 1. The transcriptional regulatory elements of AtKIX8 and AtKIX9 promoter sequences are analyzed by PlantCARE online prediction software. The results showed that auxin (IAA) response element, abscisic acid (ABA) response element, gibberellin (GA) response element and hypothermia stress response element were found in the promoter sequence of Arabidopsis thaliana. The promoters of AtKIX8 and AtKIX9 genes in Arabidopsis thaliana were cloned. The two short fragments were named pKIX8L, pKIX8SnpKIX9L and KIX9Srespectively, with the sizes of 2851 BP, 954 BP, 2982 BP and 1379 BP, respectively. The corresponding expression vectors pKIX8: GUS, PKIX8S: GUSpKIX9L: GUSpKIX9S: GUS. were successfully introduced into Agrobacterium tumefaciens GV3101.3, and pKIX8L: GuspKIX8SwarpKIX9LGUS and pKIX9Smember Gus were transformed into Arabidopsis thaliana by inflorescence. Finally, 7 pKIX8L: Gus homozygous transgenic plants and 6 pKIX8S: Gus homozygous transgenic plants, 6 pK1X9L: Gus homozygous transgenic plants, 2 pK1X9S: Gus homozygous transgenic plants, Driven by AtKIX8 gene promoter, the Gus gene was mainly expressed in the stem apical meristem region, the hypocotyls and the main veins of the young leaves, and in the leaves and stems of the 4-week flowering plants. However, no expression of Gus gene was detected in any part of AtK1X9 gene promoter. Gus positive transgenic homozygotes were selected for experiment. The quantitative results showed that Gus gene expression was high after treatment with IAA 10 渭 mol / L and low temperature (4 鈩，
本文编号：2128395
[Abstract]:The Bio ORGANS gene (LjBIO) of Leguminosae has been proved to be related to floral organ symmetry and size regulation. There are few studies on the specific function and regulation mechanism of LjBIO gene. Arabidopsis thaliana AtKIX8 and AtKIX9 genes belong to the homologous genes of LjBIO gene and belong to the KIX protein family, which are predicted to participate in the regulation of plant organ size and morphology. However, the biological function and the mechanism of the protein family are not clear. The regulation of plant organ size has a broad application prospect in production practice, which has an important guiding significance in studying the inheritance of plant morphological appearance, survival and reproduction, stress resistance and so on. Promoter is an important cis-element to regulate gene expression, which determines the timing, location and quantitative expression of specific gene. The study of plant promoter is helpful to understand the transcriptional regulation mechanism and expression pattern of the gene. It also provides clues for the study of gene function. In this study, the promoter of LjBIO homologous genes AtKIX8 and AtKIX9 were cloned and expressed in Arabidopsis thaliana, in order to reveal the mechanism of regulating organ size. It provides more clues for further elucidating the function and mechanism of LjBIO gene. The main research results obtained at present are as follows: 1. The transcriptional regulatory elements of AtKIX8 and AtKIX9 promoter sequences are analyzed by PlantCARE online prediction software. The results showed that auxin (IAA) response element, abscisic acid (ABA) response element, gibberellin (GA) response element and hypothermia stress response element were found in the promoter sequence of Arabidopsis thaliana. The promoters of AtKIX8 and AtKIX9 genes in Arabidopsis thaliana were cloned. The two short fragments were named pKIX8L, pKIX8SnpKIX9L and KIX9Srespectively, with the sizes of 2851 BP, 954 BP, 2982 BP and 1379 BP, respectively. The corresponding expression vectors pKIX8: GUS, PKIX8S: GUSpKIX9L: GUSpKIX9S: GUS. were successfully introduced into Agrobacterium tumefaciens GV3101.3, and pKIX8L: GuspKIX8SwarpKIX9LGUS and pKIX9Smember Gus were transformed into Arabidopsis thaliana by inflorescence. Finally, 7 pKIX8L: Gus homozygous transgenic plants and 6 pKIX8S: Gus homozygous transgenic plants, 6 pK1X9L: Gus homozygous transgenic plants, 2 pK1X9S: Gus homozygous transgenic plants, Driven by AtKIX8 gene promoter, the Gus gene was mainly expressed in the stem apical meristem region, the hypocotyls and the main veins of the young leaves, and in the leaves and stems of the 4-week flowering plants. However, no expression of Gus gene was detected in any part of AtK1X9 gene promoter. Gus positive transgenic homozygotes were selected for experiment. The quantitative results showed that Gus gene expression was high after treatment with IAA 10 渭 mol / L and low temperature (4 鈩，
本文编号：2128395
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