楊樹(shù)PtoDWF4在次生壁合成過(guò)程中的功能分析
發(fā)布時(shí)間：2018-08-01 16:07
【摘要】：楊樹(shù)(Populus spp.)是我國(guó)用材林、防護(hù)林和綠化林的主要樹(shù)種之一。隨著楊樹(shù)基因組測(cè)序的完成,楊樹(shù)作為木本模式植物廣泛應(yīng)用于林木分子生物學(xué)研究。然而,對(duì)于楊樹(shù)中次生壁的調(diào)控機(jī)制目前還了解不多。油菜素內(nèi)酯(Brassinosteroids,BRs)是上世紀(jì)70年代在植物中發(fā)現(xiàn)的一種類固醇激素。研究發(fā)現(xiàn),BRs在植物整個(gè)生長(zhǎng)和發(fā)育中起重要的作用,其中包括細(xì)胞伸長(zhǎng)、分裂與分化、細(xì)胞壁的合成、維管的分化、光形態(tài)建成、種子的休眠與萌發(fā)、根的發(fā)育、衰老以及生物和非生物脅迫等。目前對(duì)BRs對(duì)植物生長(zhǎng)發(fā)育影響的研究主要集中在草本植物如水稻、豌豆、煙草和擬南芥中,而在木本植物中的作用鮮有報(bào)道。在草本植物中已經(jīng)證實(shí),BRs影響次生壁的形成,然而,BRs在木本植物次生壁形成過(guò)程中的作用目前尚不清楚。DWF4是BRs合成路徑中的關(guān)鍵酶基因,它的突變將導(dǎo)致植物表現(xiàn)出矮化等特征,而外施油菜素內(nèi)酯能回復(fù)突變體表型,說(shuō)明DWF4基因在BRs生物合成及植物生長(zhǎng)發(fā)育中起著重要作用。為了探討B(tài)Rs在楊樹(shù)次生壁合成中的作用,本論文以毛白楊為研究對(duì)象,克隆了PtoDWF4,對(duì)其在調(diào)控楊樹(shù)次生壁合成途徑中的生物學(xué)功能進(jìn)行了初步研究,主要結(jié)果如下:(1)毛白楊PtoDWF4編碼一個(gè)由488個(gè)氨基酸組成的蛋白。進(jìn)化樹(shù)分析顯示,該蛋白與擬南芥和毛果楊中的DWF4序列相似性分別高達(dá)74.08%和98.57%,氨基酸序列比對(duì)發(fā)現(xiàn),PtoDWF4具有細(xì)胞色素P450家族保守的功能結(jié)構(gòu)域。(2)實(shí)時(shí)定量PCR分析組織表達(dá)譜發(fā)現(xiàn),PtoDWF4在毛白楊的根、莖、木質(zhì)部、韌皮部、幼葉和成熟葉中均有表達(dá),在莖的木質(zhì)部中表達(dá)量最高,暗示PtoDWF4可能參與了對(duì)植物次生壁中木質(zhì)部的調(diào)控。(3)過(guò)量表達(dá)PtoDWF4導(dǎo)致轉(zhuǎn)基因楊樹(shù)植株促進(jìn)了植株株高及莖直徑等指標(biāo)上升,提高了轉(zhuǎn)基因楊樹(shù)的生物量。而通過(guò)CRISPR/Cas9靶向基因編輯技術(shù)獲得的PtoDWF4突變體表現(xiàn)出植株矮小、莖變細(xì)等癥狀,表明PtoDWF4可能參與了楊樹(shù)次生壁的生物合成調(diào)控。(4)對(duì)植株的莖進(jìn)行組織切片和甲苯胺藍(lán)化學(xué)染色后觀察發(fā)現(xiàn),超表達(dá)PtoDWF4的轉(zhuǎn)基因植株莖中次生木質(zhì)部層數(shù)明顯多于野生型,而敲除PtoDWF4株系的莖中表現(xiàn)出相反表型,揭示PtoDWF4參與了楊樹(shù)莖干中次生木質(zhì)部的合成調(diào)控。(5)次生壁組分分析顯示,超表達(dá)PtoDWF4的轉(zhuǎn)基因植株中次生壁中木質(zhì)素、木聚糖、纖維素等組分均上調(diào),而敲除PtoDWF4的株系中發(fā)生了顯著下調(diào)。上述結(jié)果證明PtoDWF4在楊樹(shù)次生壁合成中起重要的調(diào)控作用。(6)熒光定量PCR檢測(cè)顯示,超表達(dá)PtoDWF4的轉(zhuǎn)基因植株中,BRs合成途徑上DWF4下游基因的相對(duì)表達(dá)量被上調(diào),而敲除PtoDWF4的植株中有一定的下降。而超量表達(dá)PtoDWF4的轉(zhuǎn)基因楊樹(shù)中木質(zhì)素、木聚糖、纖維素合成相關(guān)的結(jié)構(gòu)基因均一定程度上調(diào),相反,敲除PtoDWF4的株系中這些基因均顯著下調(diào)。在檢測(cè)次生壁合成相關(guān)的NAC/MYB轉(zhuǎn)錄因子時(shí)也發(fā)現(xiàn)了類似的結(jié)果。上述結(jié)果表明,PtoDWF4基因通過(guò)調(diào)控BRs的合成進(jìn)而影響楊樹(shù)次生壁的形成。綜上所述,本論文克隆了BR合成通路中的關(guān)鍵酶基因PtoDWF4,并通過(guò)轉(zhuǎn)基因技術(shù)、基因敲除技術(shù)、組織化學(xué)及分子實(shí)驗(yàn)等方法,初步分析了該基因在楊樹(shù)次生壁合成中的生物學(xué)功能,推測(cè)PtoDWF4可能是通過(guò)合成BRs,激活次生壁合成途徑上多個(gè)關(guān)鍵酶基因和相關(guān)轉(zhuǎn)錄因子,進(jìn)而調(diào)控楊樹(shù)次生壁的形成。本研究為楊樹(shù)材性遺傳改良提供了一定的理論基礎(chǔ)。
[Abstract]:Poplar (Populus spp.) is one of the main tree species of timber forest, protective forest and greening forest in China. With the completion of poplar genome sequencing, poplar as a woody plant is widely used in the study of forest molecular biology. However, the regulation mechanism of Yang Shuzhong secondary wall is not well understood. Brassinolide (Brassinosteroids, BRs) is not well understood. A steroid hormone found in plants in the 70s of last century. Studies have found that BRs plays an important role in the growth and development of plants, including cell elongation, division and differentiation, cell wall synthesis, vascular differentiation, light morphogenesis, seed dormancy and germination, root development, aging, and biological and abiotic stresses. Studies on the effects of BRs on plant growth and development are mainly concentrated in herbaceous plants such as rice, pea, tobacco and Arabidopsis, but the role in the woody plants is rarely reported. In herbaceous plants, BRs has been proved to affect the formation of secondary walls. However, the role of BRs in the formation of the secondary wall of woody plants is not yet clear of.DW F4 is the key enzyme gene in the BRs synthesis pathway, and its mutation will lead to dwarfing characteristics of plants, and the external application of brassinolide can restore the mutant phenotype, indicating that the DWF4 gene plays an important role in the biosynthesis of BRs and the growth and development of plants. In order to explore the role of BRs in the secondary biosynthesis of poplar, this paper is based on the study of Populus tomentosa. The main results are as follows: (1) the main results are as follows: (1) the white poplar PtoDWF4 encodes a protein composed of 488 amino acids. The phylogenetic tree analysis shows that the similarity of the protein to the DWF4 sequence of Arabidopsis and poplar is up to 74.08% and 98, respectively. .57%, the amino acid sequence alignment found that PtoDWF4 has the conservative functional domain of the cytochrome P450 family. (2) real-time quantitative PCR analysis tissue expression profiles show that PtoDWF4 is expressed in the root, stem, xylem, phloem, young leaves and mature leaves of Populus tomentosa, the highest expression in the xylem of the stem, suggesting that PtoDWF4 may be involved in the plant. The regulation of xylem in secondary wall. (3) overexpression of PtoDWF4 caused the transgenic poplar plants to increase the plant height and stem diameter, and increase the biomass of the transgenic poplar, and the PtoDWF4 mutants obtained by the CRISPR/Cas9 target gene editing technique showed that the plants were dwarf and the stem was thinner and so on, indicating that PtoDWF4 might be involved. The biosynthesis of the secondary wall of the poplar was regulated. (4) the tissue section of the stem of the plant and the chemical staining of toluidine blue showed that the number of secondary xylem in the stems of the transgenic plants with overexpression of PtoDWF4 was obviously more than that of the wild type, but the opposite phenotype was found in the stems of the PtoDWF4 plant, which revealed that PtoDWF4 was involved in the middle stem of poplar stem. Synthesis regulation of the raw xylem. (5) secondary wall component analysis showed that the components of lignin, xylan and cellulose in the secondary wall of the overexpressed PtoDWF4 were up-regulated, while the PtoDWF4 knockout was significantly down. The results showed that PtoDWF4 plays an important regulatory role in the secondary biosynthesis of poplar. (6) fluorescence quantitative P CR detection showed that in the transgenic plants with overexpression of PtoDWF4, the relative expression of the DWF4 downstream genes in the BRs synthesis pathway was up-regulated, while the plants that knocked out PtoDWF4 had a certain decline. The structural genes of the transgenic Yang Shuzhong lignin, xylan and cellulose synthesized by the overexpressed PtoDWF4 were up to a certain extent, on the contrary, Pto was knocked out of Pto. These genes were significantly down regulated in the DWF4 strains. Similar results were found when detecting the NAC/MYB transcription factors related to the secondary wall synthesis. The results showed that the PtoDWF4 gene affected the formation of the secondary wall of poplar by regulating the synthesis of BRs. In this paper, the key enzyme gene PtoDWF4 in the BR synthesis pathway was cloned. The biological function of the gene in the secondary wall synthesis of poplar was preliminarily analyzed by gene knockout technique, gene knockout technique, histochemistry and molecular experiments. It is suggested that PtoDWF4 may activate multiple key enzyme genes and phase transcription factors on the secondary wall synthesis pathway by synthesizing BRs, and then regulate the formation of the secondary wall of poplar. The study provided a theoretical basis for genetic improvement of poplar wood properties.
【學(xué)位授予單位】：西南大學(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)：2158111
[Abstract]:Poplar (Populus spp.) is one of the main tree species of timber forest, protective forest and greening forest in China. With the completion of poplar genome sequencing, poplar as a woody plant is widely used in the study of forest molecular biology. However, the regulation mechanism of Yang Shuzhong secondary wall is not well understood. Brassinolide (Brassinosteroids, BRs) is not well understood. A steroid hormone found in plants in the 70s of last century. Studies have found that BRs plays an important role in the growth and development of plants, including cell elongation, division and differentiation, cell wall synthesis, vascular differentiation, light morphogenesis, seed dormancy and germination, root development, aging, and biological and abiotic stresses. Studies on the effects of BRs on plant growth and development are mainly concentrated in herbaceous plants such as rice, pea, tobacco and Arabidopsis, but the role in the woody plants is rarely reported. In herbaceous plants, BRs has been proved to affect the formation of secondary walls. However, the role of BRs in the formation of the secondary wall of woody plants is not yet clear of.DW F4 is the key enzyme gene in the BRs synthesis pathway, and its mutation will lead to dwarfing characteristics of plants, and the external application of brassinolide can restore the mutant phenotype, indicating that the DWF4 gene plays an important role in the biosynthesis of BRs and the growth and development of plants. In order to explore the role of BRs in the secondary biosynthesis of poplar, this paper is based on the study of Populus tomentosa. The main results are as follows: (1) the main results are as follows: (1) the white poplar PtoDWF4 encodes a protein composed of 488 amino acids. The phylogenetic tree analysis shows that the similarity of the protein to the DWF4 sequence of Arabidopsis and poplar is up to 74.08% and 98, respectively. .57%, the amino acid sequence alignment found that PtoDWF4 has the conservative functional domain of the cytochrome P450 family. (2) real-time quantitative PCR analysis tissue expression profiles show that PtoDWF4 is expressed in the root, stem, xylem, phloem, young leaves and mature leaves of Populus tomentosa, the highest expression in the xylem of the stem, suggesting that PtoDWF4 may be involved in the plant. The regulation of xylem in secondary wall. (3) overexpression of PtoDWF4 caused the transgenic poplar plants to increase the plant height and stem diameter, and increase the biomass of the transgenic poplar, and the PtoDWF4 mutants obtained by the CRISPR/Cas9 target gene editing technique showed that the plants were dwarf and the stem was thinner and so on, indicating that PtoDWF4 might be involved. The biosynthesis of the secondary wall of the poplar was regulated. (4) the tissue section of the stem of the plant and the chemical staining of toluidine blue showed that the number of secondary xylem in the stems of the transgenic plants with overexpression of PtoDWF4 was obviously more than that of the wild type, but the opposite phenotype was found in the stems of the PtoDWF4 plant, which revealed that PtoDWF4 was involved in the middle stem of poplar stem. Synthesis regulation of the raw xylem. (5) secondary wall component analysis showed that the components of lignin, xylan and cellulose in the secondary wall of the overexpressed PtoDWF4 were up-regulated, while the PtoDWF4 knockout was significantly down. The results showed that PtoDWF4 plays an important regulatory role in the secondary biosynthesis of poplar. (6) fluorescence quantitative P CR detection showed that in the transgenic plants with overexpression of PtoDWF4, the relative expression of the DWF4 downstream genes in the BRs synthesis pathway was up-regulated, while the plants that knocked out PtoDWF4 had a certain decline. The structural genes of the transgenic Yang Shuzhong lignin, xylan and cellulose synthesized by the overexpressed PtoDWF4 were up to a certain extent, on the contrary, Pto was knocked out of Pto. These genes were significantly down regulated in the DWF4 strains. Similar results were found when detecting the NAC/MYB transcription factors related to the secondary wall synthesis. The results showed that the PtoDWF4 gene affected the formation of the secondary wall of poplar by regulating the synthesis of BRs. In this paper, the key enzyme gene PtoDWF4 in the BR synthesis pathway was cloned. The biological function of the gene in the secondary wall synthesis of poplar was preliminarily analyzed by gene knockout technique, gene knockout technique, histochemistry and molecular experiments. It is suggested that PtoDWF4 may activate multiple key enzyme genes and phase transcription factors on the secondary wall synthesis pathway by synthesizing BRs, and then regulate the formation of the secondary wall of poplar. The study provided a theoretical basis for genetic improvement of poplar wood properties.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2
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