擬南芥中LjBIO同源基因AtKIX8和AtKIX9啟動(dòng)子的克隆及表達(dá)分析
發(fā)布時(shí)間：2018-07-17 01:19
【摘要】：豆科植物百脈根的BIO ORGANS基因(LjBIO)已經(jīng)被證實(shí)與花器官對(duì)稱性和大小調(diào)控有關(guān),目前有關(guān)于LjBIO基因具體功能和調(diào)控機(jī)制的研究報(bào)告很少。擬南芥AtKIX8和AtKIX9基因與百脈根LjBIO基因?qū)偻椿?編碼產(chǎn)物屬于KIX蛋白家族成員,被預(yù)測(cè)參與了植物器官大小及形態(tài)調(diào)控,但該蛋白家族的生物學(xué)功能及其作用機(jī)理尚不清楚。調(diào)控植物的器官大小在生產(chǎn)實(shí)踐中有廣泛的應(yīng)用前景,對(duì)研究植物形態(tài)外觀、生存繁殖、脅迫抗性等品質(zhì)的遺傳有重要指導(dǎo)意義。啟動(dòng)子是調(diào)控基因表達(dá)的重要順式元件,決定著特定基因的定時(shí)、定位、定量表達(dá),對(duì)植物啟動(dòng)子的研究有助于了解該基因的轉(zhuǎn)錄調(diào)控機(jī)制及其表達(dá)模式,并為研究基因的功能提供線索。本研究以模式植物擬南芥為研究材料,對(duì)LjBIO同源基因AtKIX8和AtKIX9的啟動(dòng)子進(jìn)行了克隆和表達(dá)分析研究,期望揭示該類基因調(diào)節(jié)器官大小的作用機(jī)制,并為進(jìn)一步闡明LjBIO基因的功能及機(jī)理提供更多的線索�，F(xiàn)階段取得的主要研究成果如下：1、利用PlantCARE在線預(yù)測(cè)軟件對(duì)AtKIX8和AtKIX9啟動(dòng)子序列進(jìn)行轉(zhuǎn)錄調(diào)控元件分析。分析結(jié)果表明,它們的啟動(dòng)子序列中含有生長(zhǎng)素(IAA)響應(yīng)元件、脫落酸(ABA)響應(yīng)元件、赤霉素(GA)響應(yīng)元件、低溫脅迫響應(yīng)元件等元件。2、克隆了擬南芥AtKIX8和AtKIX9基因啟動(dòng)子各長(zhǎng)、短兩個(gè)片段,分別命名為pKIX8L、pKIX8S、pKIX9L和KIX9S,大小分別為2851 bp、954 bp、2982bp和1379 bp。并構(gòu)建相應(yīng)接GUS報(bào)告基因的表達(dá)載體pKIX8:GUS、 PKIX8S:GUS、pKIX9L:GUS、pKIX9S:GUS,成功將這4個(gè)表達(dá)載體導(dǎo)入根癌農(nóng)桿菌GV3101。3、通過(guò)花序侵染法將pKIX8L:GUS、pKIX8S:GUS、pKIX9L:GUS和pKIX9S:GUS這4個(gè)表達(dá)載體遺傳轉(zhuǎn)化擬南芥,用Hyg篩選轉(zhuǎn)基因陽(yáng)性植株,最終獲得7株轉(zhuǎn)pKIX8L:GUS的純合轉(zhuǎn)基因植株,6株轉(zhuǎn)pKIX8S:GUS的純合轉(zhuǎn)基因植株,6株轉(zhuǎn)pK1X9L:GUS的純合轉(zhuǎn)基因植株,2株轉(zhuǎn)pK1X9S:GUS的純合轉(zhuǎn)基因植株。4、GUS染色結(jié)果顯示,在AtKIX8基因啟動(dòng)子的驅(qū)動(dòng)下,GUS基因主要集中在10天幼苗的莖尖分生組織區(qū)、下胚軸中柱以及幼葉的主脈等維管系統(tǒng)中,以及4周開(kāi)花植物的葉片主脈和莖中有強(qiáng)表達(dá)。而在AtK1X9基因啟動(dòng)子驅(qū)動(dòng)下,未檢測(cè)到GUS基因在任何部位有表達(dá)。5、選取GUS染色陽(yáng)性的轉(zhuǎn)基因純合體進(jìn)行實(shí)驗(yàn),定量結(jié)果顯示在生長(zhǎng)素(IAA 10μmol/L)和低溫(4℃)處理后GUS基因的表達(dá)量高,而在脫落酸(ABA 200μmol/L)和赤霉素(GA 50μmol/L)處理后GUS基因表達(dá)量并無(wú)顯著差異。
[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，
本文編號(hào)：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，
本文編號(hào)：2128395
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