PUBX1和PUBX2基因?qū)M南芥器官發(fā)育的調(diào)控
發(fā)布時(shí)間：2019-06-22 15:19
【摘要】：植物器官的大小的調(diào)控是一個(gè)基本的發(fā)育生物學(xué)過(guò)程,其生長(zhǎng)受發(fā)育遺傳程序和環(huán)境因素的影響,其研究是植物發(fā)育生物學(xué)領(lǐng)域的重要前沿和研究熱點(diǎn)。植物不同物種之間器官大小差異巨大,而特定物種的器官最終大小卻是一定的,表明植物器官生長(zhǎng)到最終大小受到嚴(yán)密的遺傳控制。器官生長(zhǎng)受到的時(shí)空調(diào)節(jié),主要分為細(xì)胞分裂和細(xì)胞體積擴(kuò)增兩個(gè)不同的過(guò)程。本項(xiàng)目以模式植物擬南芥為研究材料,通過(guò)分子生物學(xué),細(xì)胞生物學(xué),生物化學(xué)等綜合研究手段,對(duì)調(diào)節(jié)擬南芥器官發(fā)育生長(zhǎng)的重要基因PUBX1及其同源基因PUBX2的功能進(jìn)行研究,解析高等植物器官大小決定的遺傳和分子機(jī)制,為植物生長(zhǎng)發(fā)育的遺傳改良提供理論依據(jù)。本研究通過(guò)對(duì)野生型,pubx1,pubx2單突變體以及pubx1 pubx2雙突變體的花瓣和葉片的長(zhǎng)度、寬度、表面積以及莖的直徑的表型進(jìn)行拍照觀察并測(cè)量統(tǒng)計(jì),發(fā)現(xiàn)單突變體與WT相比無(wú)明顯變化,而雙突變體的表型非常明顯,因此PUBX1和PUBX2是有功能冗余的影響器官大小的基因。我們利用Gateway技術(shù)構(gòu)建了PUBX1和PUBX2基因的過(guò)表達(dá)植株,通過(guò)RT—qPCR對(duì)其表達(dá)量分析可知,PUB基因表達(dá)量與植株器官大小呈負(fù)相關(guān),表達(dá)量越高的植株器官越小。我們同時(shí)構(gòu)建了△PUBX1和△PUBX2基因的過(guò)表達(dá)植株作為對(duì)照,RT—qPCR結(jié)果可得表達(dá)量明顯上調(diào)的刪除U-box結(jié)構(gòu)域的植株表型與WT相比無(wú)顯著變化,進(jìn)一步驗(yàn)證了U-box結(jié)構(gòu)域?qū)τ赑UBX1和PUBX2基因的酶活是必需的。為了進(jìn)一步了解PUBX1和PUBX2基因的作用機(jī)制,我們構(gòu)建了表達(dá)PUB基因啟動(dòng)子及全長(zhǎng)蛋白與GUS報(bào)告基因的融合蛋白的轉(zhuǎn)基因植株,篩選陽(yáng)性植株進(jìn)行GUS組織染色來(lái)研究PUB基因的表達(dá)模式,發(fā)現(xiàn)其在成熟的花瓣中表達(dá)量遠(yuǎn)遠(yuǎn)大于幼嫩的花瓣中。同時(shí)我們測(cè)量統(tǒng)計(jì)并繪制了花瓣的生長(zhǎng)曲線,得出PUBX1和PUBX2基因是通過(guò)調(diào)控細(xì)胞分裂持續(xù)時(shí)間進(jìn)而影響植株器官發(fā)育的大小。本研究通過(guò)對(duì)植物器官大小調(diào)控機(jī)制的探究,為作物的遺傳改良提供理論依據(jù),對(duì)植物器官大小調(diào)控的機(jī)制研究將有助于提高作物產(chǎn)量,解決糧食危機(jī)有深遠(yuǎn)的現(xiàn)實(shí)意義。
[Abstract]:The regulation of plant organ size is a basic developmental biological process, its growth is affected by developmental genetic procedures and environmental factors, and its research is an important frontier and research focus in the field of plant developmental biology. There are great differences in organ size among different species of plants, but the final size of organs in specific species is certain, indicating that the growth of plant organs to the final size is closely controlled by heredity. The temporal and spatial regulation of organ growth is mainly divided into two different processes: cell division and cell volume amplification. In this project, Arabidopsis thaliana, a model plant, was used as the research material to study the function of the important gene PUBX1 and its homologous gene PUBX2, which regulate the organ development and growth of Arabidopsis thaliana by means of molecular biology, cell biology and biochemistry. The genetic and molecular mechanism of organ size determination in higher plants was analyzed, which provided a theoretical basis for the genetic improvement of plant growth and development. In this study, the length, width, surface area and stem diameter of petals and leaves of wild type, pubx1,pubx2 single mutant and pubx1 pubx2 double mutant were observed and measured. It was found that there was no significant change in the phenotype of single mutant compared with WT, but the phenotype of double mutant was very obvious, so PUBX1 and PUBX2 were genes with functional redundancy affecting organ size. The overexpression of PUBX1 and PUBX2 genes was constructed by Gateway. RT-qPCR analysis showed that the expression of PUB gene was negatively correlated with plant organ size, and the higher the expression was, the smaller the plant organs were. At the same time, we constructed the overexpression plants of PUBX1 and PUBX2 genes as control. The results of RT-qPCR showed that the phenotypes of plants deleted U-box domain with up-regulated expression had no significant change compared with WT, which further verified that U-box domain was necessary for the enzyme activity of PUBX1 and PUBX2 genes. In order to further understand the mechanism of PUBX1 and PUBX2 gene, we constructed transgenic plants expressing PUB gene promoter and fusion protein of full-length protein and GUS reporter gene. Positive plants were screened for GUS tissue staining to study the expression pattern of PUB gene. It was found that the expression of PUB gene in mature petals was much higher than that in young petals. At the same time, we measured and plotted the growth curve of petals, and it was concluded that PUBX1 and PUBX2 genes affected the development of plant organs by regulating the duration of cell division. Through the study of the regulation mechanism of plant organ size, this study provides a theoretical basis for the genetic improvement of crops, and the study of the mechanism of plant organ size regulation will be helpful to improve crop yield and solve the food crisis.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2
本文編號(hào)：2504703
[Abstract]:The regulation of plant organ size is a basic developmental biological process, its growth is affected by developmental genetic procedures and environmental factors, and its research is an important frontier and research focus in the field of plant developmental biology. There are great differences in organ size among different species of plants, but the final size of organs in specific species is certain, indicating that the growth of plant organs to the final size is closely controlled by heredity. The temporal and spatial regulation of organ growth is mainly divided into two different processes: cell division and cell volume amplification. In this project, Arabidopsis thaliana, a model plant, was used as the research material to study the function of the important gene PUBX1 and its homologous gene PUBX2, which regulate the organ development and growth of Arabidopsis thaliana by means of molecular biology, cell biology and biochemistry. The genetic and molecular mechanism of organ size determination in higher plants was analyzed, which provided a theoretical basis for the genetic improvement of plant growth and development. In this study, the length, width, surface area and stem diameter of petals and leaves of wild type, pubx1,pubx2 single mutant and pubx1 pubx2 double mutant were observed and measured. It was found that there was no significant change in the phenotype of single mutant compared with WT, but the phenotype of double mutant was very obvious, so PUBX1 and PUBX2 were genes with functional redundancy affecting organ size. The overexpression of PUBX1 and PUBX2 genes was constructed by Gateway. RT-qPCR analysis showed that the expression of PUB gene was negatively correlated with plant organ size, and the higher the expression was, the smaller the plant organs were. At the same time, we constructed the overexpression plants of PUBX1 and PUBX2 genes as control. The results of RT-qPCR showed that the phenotypes of plants deleted U-box domain with up-regulated expression had no significant change compared with WT, which further verified that U-box domain was necessary for the enzyme activity of PUBX1 and PUBX2 genes. In order to further understand the mechanism of PUBX1 and PUBX2 gene, we constructed transgenic plants expressing PUB gene promoter and fusion protein of full-length protein and GUS reporter gene. Positive plants were screened for GUS tissue staining to study the expression pattern of PUB gene. It was found that the expression of PUB gene in mature petals was much higher than that in young petals. At the same time, we measured and plotted the growth curve of petals, and it was concluded that PUBX1 and PUBX2 genes affected the development of plant organs by regulating the duration of cell division. Through the study of the regulation mechanism of plant organ size, this study provides a theoretical basis for the genetic improvement of crops, and the study of the mechanism of plant organ size regulation will be helpful to improve crop yield and solve the food crisis.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
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