杨树PtoDWF4在次生壁合成过程中的功能分析

发布时间：2018-08-01 16:07

【摘要】：杨树(Populus spp.)是我国用材林、防护林和绿化林的主要树种之一。随着杨树基因组测序的完成,杨树作为木本模式植物广泛应用于林木分子生物学研究。然而,对于杨树中次生壁的调控机制目前还了解不多。油菜素内酯(Brassinosteroids,BRs)是上世纪70年代在植物中发现的一种类固醇激素。研究发现,BRs在植物整个生长和发育中起重要的作用,其中包括细胞伸长、分裂与分化、细胞壁的合成、维管的分化、光形态建成、种子的休眠与萌发、根的发育、衰老以及生物和非生物胁迫等。目前对BRs对植物生长发育影响的研究主要集中在草本植物如水稻、豌豆、烟草和拟南芥中,而在木本植物中的作用鲜有报道。在草本植物中已经证实,BRs影响次生壁的形成,然而,BRs在木本植物次生壁形成过程中的作用目前尚不清楚。DWF4是BRs合成路径中的关键酶基因,它的突变将导致植物表现出矮化等特征,而外施油菜素内酯能回复突变体表型,说明DWF4基因在BRs生物合成及植物生长发育中起着重要作用。为了探讨BRs在杨树次生壁合成中的作用,本论文以毛白杨为研究对象,克隆了PtoDWF4,对其在调控杨树次生壁合成途径中的生物学功能进行了初步研究,主要结果如下:(1)毛白杨PtoDWF4编码一个由488个氨基酸组成的蛋白。进化树分析显示,该蛋白与拟南芥和毛果杨中的DWF4序列相似性分别高达74.08%和98.57%,氨基酸序列比对发现,PtoDWF4具有细胞色素P450家族保守的功能结构域。(2)实时定量PCR分析组织表达谱发现,PtoDWF4在毛白杨的根、茎、木质部、韧皮部、幼叶和成熟叶中均有表达,在茎的木质部中表达量最高,暗示PtoDWF4可能参与了对植物次生壁中木质部的调控。(3)过量表达PtoDWF4导致转基因杨树植株促进了植株株高及茎直径等指标上升,提高了转基因杨树的生物量。而通过CRISPR/Cas9靶向基因编辑技术获得的PtoDWF4突变体表现出植株矮小、茎变细等症状,表明PtoDWF4可能参与了杨树次生壁的生物合成调控。(4)对植株的茎进行组织切片和甲苯胺蓝化学染色后观察发现,超表达PtoDWF4的转基因植株茎中次生木质部层数明显多于野生型,而敲除PtoDWF4株系的茎中表现出相反表型,揭示PtoDWF4参与了杨树茎干中次生木质部的合成调控。(5)次生壁组分分析显示,超表达PtoDWF4的转基因植株中次生壁中木质素、木聚糖、纤维素等组分均上调,而敲除PtoDWF4的株系中发生了显著下调。上述结果证明PtoDWF4在杨树次生壁合成中起重要的调控作用。(6)荧光定量PCR检测显示,超表达PtoDWF4的转基因植株中,BRs合成途径上DWF4下游基因的相对表达量被上调,而敲除PtoDWF4的植株中有一定的下降。而超量表达PtoDWF4的转基因杨树中木质素、木聚糖、纤维素合成相关的结构基因均一定程度上调,相反,敲除PtoDWF4的株系中这些基因均显著下调。在检测次生壁合成相关的NAC/MYB转录因子时也发现了类似的结果。上述结果表明,PtoDWF4基因通过调控BRs的合成进而影响杨树次生壁的形成。综上所述,本论文克隆了BR合成通路中的关键酶基因PtoDWF4,并通过转基因技术、基因敲除技术、组织化学及分子实验等方法,初步分析了该基因在杨树次生壁合成中的生物学功能,推测PtoDWF4可能是通过合成BRs,激活次生壁合成途径上多个关键酶基因和相关转录因子,进而调控杨树次生壁的形成。本研究为杨树材性遗传改良提供了一定的理论基础。
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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