杨树糖转运体SWEETs和SUT4的功能分析

发布时间：2018-06-23 09:11

  本文选题：杨树 + SWEET基因家族　； 参考：《中国林业科学研究院》2016年博士论文

【摘要】：在高等植物中,糖作为主要的储能物质、重要的信号分子、渗透调节物质及其它有机物合成的骨架,在植物的整个生命周期中发挥重要作用。植物糖转运体在糖运输的多个过程中发挥重要作用,包括通过维管组织进行的长距离运输、细胞到细胞间的运输和细胞内的运输,对植物生长发育至关重要。尽管目前拟南芥和水稻中的糖转运体已进行了部分研究,但在木本植物杨树中尚未进行系统研究。木本植物与一年生草本植物相比具有多个特殊的发育过程,如木材的形成需要从光合组织获得碳源。因此,研究杨树中糖转运体的功能有助于揭示树木碳分配的机制,并为通过控制碳源分配调控木材发育提供理论基础。本研究对杨树SWEET基因家族进行了鉴定和系统分析,包括进化关系、表达模式和糖转运特性,并通过转基因对PtSWEET5、PtSWEET7和PtSWEET17a在杨树生长发育中的作用进行了分析。另外,对杨树SUT基因家族的PtSUT4基因在次生生长中的作用进行了进一步研究。研究结果如下:1.本研究共鉴定出27个杨树SWEET基因,是拟南芥SWEET家族基因数量的1.59倍。进化分析发现杨树SWEET基因家族可以分为4个Clade,并且每个Clade中的基因结构和保守基序组成相对保守。片段复制和串联重复共同造成了杨树SWEET基因家族的扩张。2.为研究杨树SWEETs蛋白的亚细胞定位,共选取了9个代表不同Clade的成员,构建其与YFP的N-融合的载体,并瞬时转化烟草叶片。共聚焦显微镜分析显示,PtSWEET4-YFP、PtSWEET7-YFP和PtSWEET15b-YFP定位在细胞质膜,PtSWEET17b-YFP和PtSWEET17d-YFP的YFP信号在液泡膜上被检测到。PtSWEET2c-YFP、PtSWEET3b-YFP和PtSWEET17a-YFP的信号位于内质网,PtSWEET16a-YFP的信号位于高尔基体。这些结果说明,杨树SWEETs可能在细胞间或细胞内糖的运输和分配中均发挥重要作用。为研究PtSWEETs的转运功能,共选取12个代表不同Clade的成员,分别转化己糖吸收缺陷型酵母突变体EBY.VW4000,并进行糖转运体功能的研究。互补生长分析发现,PtSWEET4、PtSWEET5和PtSWEET7在酵母细胞中具有葡萄糖、甘露糖和半乳糖的转运活性,ptsweet15a具有运输半乳糖的能力。这说明ptsweets在杨树中可能具有运输多种糖的能力。3.实时荧光定量pcr分析发现ptsweets具有多样的组织表达模式。ptsweet1a和ptsweet1b主要在根中表达,ptsweet2c、3b和10a主要在成熟叶片中表达,ptsweet5、7、10b和10c主要在韧皮部中表达,ptsweet15b在木质部中特异表达,ptsweet4、5和15a主要在雄花中表达,ptsweet9和ptsweet11只在雄花和雌花中表达。gus组织染色分析发现,ptsweet2c在成熟叶片中的表达量高于其它组织,ptsweet7在韧皮部中特异表达,ptsweet17a在韧皮部、木质部、射线细胞和根中高表达,ptsweet15b在木质部特异表达。这些结果与定量pcr分析结果一致。组织表达模式的多样性,说明ptsweets基因可能参与了杨树的多个生物学过程,在茎中高表达的基因可能参与了木质部发育过程中糖的转运。4.为研究ptsweet5、ptsweet7和ptsweet17a的功能,本研究分别创制了ptsweet5、ptsweet7和ptsweet17a的超表达转基因杨树。超表达ptsweet5促进了转基因杨树木质部和韧皮部的发育,加速了叶片和根的衰老,并抑制了杨树生长。pi/fda双荧光染色发现,ptsweet5超表达株系根细胞的活力降低。与此相反,超表达ptsweet7和ptsweet17a均促进了杨树生长和木质部发育,说明两个基因在杨树次生生长中均发挥重要作用。5.亚细胞定位和qrt-pcr分析表明,ptsut4基因编码液泡膜定位的蔗糖转运蛋白,并在各组织中表达,在成熟叶、茎、韧皮部和花中具有较高表达。ptsut4超表达转基因株系(s1和s12)的株高分别增加了22%和17%,地径分别增加了9%和7%。另外,2个超表达株系第七节间木质部宽度与对照株系相比,分别增加了32%和21%。超表达株系的净光合速率显著高于对照,分别高出24%和21%。2个株系的光合作用增强,说明ptsut4可能通过促进蔗糖在源端的装载和在库端的卸载而对光合作用产生正反馈效应。光合作用的增强和茎中蔗糖卸载效率的增加,促进了杨树的高生长和径向生长(次生木质部发育)。本研究分析了27个ptsweet基因的进化和部分糖转运特征,其多样的表达模式、不同的亚细胞定位和糖转运活性暗示其可能参与木本植物的多个发育过程。进一步通过转基因技术发现了ptsweet5、ptsweet7、ptsweet17a和ptsut4在次生生长中发挥重要作用。这些结果为深入研究杨树糖转运体的功能和调控提供了线索,并为林木速生或材性改良提供重要的理论和技术基础。
[Abstract]:In higher plants, sugar is the main energy storage substance, important signal molecules, osmotic substances and other organic compounds, which play an important role in the whole life cycle of plants. Plant sugar transporters play an important role in the process of sugar transport, including long distance transport through vascular tissue, cells Transportation and transport within cells are essential for the growth and development of plants. Although some studies have been carried out on sugar transporters in Arabidopsis and rice, Yang Shuzhong has not been systematically studied in woody plants. The woody plants have many special developmental processes compared with annual herbs, such as the formation of wood. To obtain carbon sources from photosynthetic tissues, the study of the function of sugar transporters in poplar can help to reveal the mechanism of carbon distribution in trees and provide a theoretical basis for controlling the development of wood by controlling the distribution of carbon sources. The SWEET gene family of poplar was identified and systematically analyzed, including evolutionary relationships, expression patterns and sugar transport characteristics, The role of PtSWEET5, PtSWEET7 and PtSWEET17a in the growth and development of poplar was analyzed by transgene. In addition, the role of PtSUT4 gene in the SUT gene family of poplar in secondary growth was further studied. The results were as follows: 1. a total of 27 Populus tree SWEET genes were identified, the number of SWEET family genes in Arabidopsis thaliana. The evolutionary analysis found that the SWEET gene family of poplar can be divided into 4 Clade, and the composition of the gene structure and the conserved sequence in each Clade is relatively conservative. Fragment replication and tandem repeat cause the expansion.2. of the SWEET gene family of poplar to study the subcellular localization of the SWEETs protein of poplar, and a total of 9 different Clade are selected to represent different Clade. A confocal microscope analysis showed that PtSWEET4-YFP, PtSWEET7-YFP and PtSWEET15b-YFP were located in the cell plasmalemma, and the YFP signals of PtSWEET17b-YFP and PtSWEET17d-YFP were detected on the vacuolar membrane for.PtSWEET2c-YFP, PtSWEET3b-YFP and PtSWEET17a-YFP signals located in the vacuolar membrane. The signal of the endoplasmic reticulum, PtSWEET16a-YFP, is located in the Golgi body. These results suggest that poplar SWEETs may play an important role in the transport and distribution of sugar in both cells and cells. In order to study the transport function of PtSWEETs, 12 members of different Clade were selected to convert the hexose absorption defective yeast mutant EBY.VW4000 respectively. The complementary growth analysis found that PtSWEET4, PtSWEET5 and PtSWEET7 have the transport activity of glucose, mannose and galactose in the yeast cells, and ptsweet15a has the ability to transport galactose. This shows that ptsweets may have the ability to transport multiple sugars in Yang Shuzhong by.3. real-time quantitative PCR analysis of PCR and found Pt Sweets has a variety of tissue expression patterns,.Ptsweet1a and ptsweet1b, mainly expressed in roots. Ptsweet2c, 3b and 10A are mainly expressed in mature leaves. Ptsweet5,7,10b and 10C are mainly expressed in phloem. Ptsweet15b is specifically expressed in the xylem. Ptsweet4,5 and 15A are mainly expressed in male flowers. Ptsweet9 and females are only in male and female flowers. The expression of.Gus tissue staining in the flowers showed that the expression of ptsweet2c in mature leaves was higher than that of other tissues, ptsweet7 was expressed specifically in phloem, ptsweet17a was highly expressed in phloem, xylem, ray cells and roots, and ptsweet15b was expressed in xylem. These results were consistent with quantitative PCR analysis. Tissue expression pattern was consistent. Diversity suggests that the ptsweets gene may be involved in many biological processes in poplar. The gene expressed in the stem may participate in the transport of sugar in the process of xylem development by.4. to study the functions of ptsweet5, ptsweet7 and ptsweet17a. The overexpression of ptsweet5, ptsweet7 and ptsweet17a in transgenic poplars. Sweet5 promoted the development of xylem and phloem in transgenic poplar, accelerated the senescence of leaves and roots, and inhibited the growth of.Pi/fda double fluorescent staining of poplar growth. The activity of ptsweet5 overexpressed root cells decreased. On the contrary, the overexpression of ptsweet7 and ptsweet17a promoted the growth of poplar and the development of xylem, indicating two genes. The important role of.5. subcellular localization and qRT-PCR analysis in the secondary growth of poplar showed that the ptsut4 gene encoded the sucrose transporter in the vacuolar membrane and expressed in the tissues. The height of the transgenic lines (S1 and S12) with high expression of.Ptsut4 overexpression in the mature leaves, stems, phloem and flowers increased by 22% and 17%, respectively. The diameter increased by 9% and 7%., respectively. Compared with the control line, the width of the seventh internode xylem of the 2 overexpressed strains increased by 32% and 21%., respectively. The net photosynthetic rate of the seventh strains was significantly higher than that of the control. The photosynthesis of 24% and 21%.2 strains increased respectively, indicating that ptsut4 could promote the loading of sucrose at the source end and the end of the library. The increase of photosynthesis and the increase in sucrose unloading efficiency in the stem promote the high growth and radial growth of the poplar (secondary xylem Development). This study analyzed the evolution and partial sugar transport characteristics of the 27 ptsweet genes, the diversity of expression patterns, different subcellular localization and sugar. Transport activity suggests that it may be involved in multiple developmental processes in woody plants. Ptsweet5, ptsweet7, ptsweet17a and ptsut4 have been found to play an important role in secondary growth through transgenic technology. These results provide clues for further study of the function and regulation of poplar sugar transporters, and provide a rapid or material improvement for forest trees. Important theoretical and technical basis.
【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S792.11
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