本文選題：楊樹(shù) + SWEET基因家族��； 參考：《中國(guó)林業(yè)科學(xué)研究院》2016年博士論文

【摘要】：在高等植物中,糖作為主要的儲(chǔ)能物質(zhì)、重要的信號(hào)分子、滲透調(diào)節(jié)物質(zhì)及其它有機(jī)物合成的骨架,在植物的整個(gè)生命周期中發(fā)揮重要作用。植物糖轉(zhuǎn)運(yùn)體在糖運(yùn)輸?shù)亩鄠�(gè)過(guò)程中發(fā)揮重要作用,包括通過(guò)維管組織進(jìn)行的長(zhǎng)距離運(yùn)輸、細(xì)胞到細(xì)胞間的運(yùn)輸和細(xì)胞內(nèi)的運(yùn)輸,對(duì)植物生長(zhǎng)發(fā)育至關(guān)重要。盡管目前擬南芥和水稻中的糖轉(zhuǎn)運(yùn)體已進(jìn)行了部分研究,但在木本植物楊樹(shù)中尚未進(jìn)行系統(tǒng)研究。木本植物與一年生草本植物相比具有多個(gè)特殊的發(fā)育過(guò)程,如木材的形成需要從光合組織獲得碳源。因此,研究楊樹(shù)中糖轉(zhuǎn)運(yùn)體的功能有助于揭示樹(shù)木碳分配的機(jī)制,并為通過(guò)控制碳源分配調(diào)控木材發(fā)育提供理論基礎(chǔ)。本研究對(duì)楊樹(shù)SWEET基因家族進(jìn)行了鑒定和系統(tǒng)分析,包括進(jìn)化關(guān)系、表達(dá)模式和糖轉(zhuǎn)運(yùn)特性,并通過(guò)轉(zhuǎn)基因?qū)tSWEET5、PtSWEET7和PtSWEET17a在楊樹(shù)生長(zhǎng)發(fā)育中的作用進(jìn)行了分析。另外,對(duì)楊樹(shù)SUT基因家族的PtSUT4基因在次生生長(zhǎng)中的作用進(jìn)行了進(jìn)一步研究。研究結(jié)果如下:1.本研究共鑒定出27個(gè)楊樹(shù)SWEET基因,是擬南芥SWEET家族基因數(shù)量的1.59倍。進(jìn)化分析發(fā)現(xiàn)楊樹(shù)SWEET基因家族可以分為4個(gè)Clade,并且每個(gè)Clade中的基因結(jié)構(gòu)和保守基序組成相對(duì)保守。片段復(fù)制和串聯(lián)重復(fù)共同造成了楊樹(shù)SWEET基因家族的擴(kuò)張。2.為研究楊樹(shù)SWEETs蛋白的亞細(xì)胞定位,共選取了9個(gè)代表不同Clade的成員,構(gòu)建其與YFP的N-融合的載體,并瞬時(shí)轉(zhuǎn)化煙草葉片。共聚焦顯微鏡分析顯示,PtSWEET4-YFP、PtSWEET7-YFP和PtSWEET15b-YFP定位在細(xì)胞質(zhì)膜,PtSWEET17b-YFP和PtSWEET17d-YFP的YFP信號(hào)在液泡膜上被檢測(cè)到。PtSWEET2c-YFP、PtSWEET3b-YFP和PtSWEET17a-YFP的信號(hào)位于內(nèi)質(zhì)網(wǎng),PtSWEET16a-YFP的信號(hào)位于高爾基體。這些結(jié)果說(shuō)明,楊樹(shù)SWEETs可能在細(xì)胞間或細(xì)胞內(nèi)糖的運(yùn)輸和分配中均發(fā)揮重要作用。為研究PtSWEETs的轉(zhuǎn)運(yùn)功能,共選取12個(gè)代表不同Clade的成員,分別轉(zhuǎn)化己糖吸收缺陷型酵母突變體EBY.VW4000,并進(jìn)行糖轉(zhuǎn)運(yùn)體功能的研究�；パa(bǔ)生長(zhǎng)分析發(fā)現(xiàn),PtSWEET4、PtSWEET5和PtSWEET7在酵母細(xì)胞中具有葡萄糖、甘露糖和半乳糖的轉(zhuǎn)運(yùn)活性,ptsweet15a具有運(yùn)輸半乳糖的能力。這說(shuō)明ptsweets在楊樹(shù)中可能具有運(yùn)輸多種糖的能力。3.實(shí)時(shí)熒光定量pcr分析發(fā)現(xiàn)ptsweets具有多樣的組織表達(dá)模式。ptsweet1a和ptsweet1b主要在根中表達(dá),ptsweet2c、3b和10a主要在成熟葉片中表達(dá),ptsweet5、7、10b和10c主要在韌皮部中表達(dá),ptsweet15b在木質(zhì)部中特異表達(dá),ptsweet4、5和15a主要在雄花中表達(dá),ptsweet9和ptsweet11只在雄花和雌花中表達(dá)。gus組織染色分析發(fā)現(xiàn),ptsweet2c在成熟葉片中的表達(dá)量高于其它組織,ptsweet7在韌皮部中特異表達(dá),ptsweet17a在韌皮部、木質(zhì)部、射線(xiàn)細(xì)胞和根中高表達(dá),ptsweet15b在木質(zhì)部特異表達(dá)。這些結(jié)果與定量pcr分析結(jié)果一致。組織表達(dá)模式的多樣性,說(shuō)明ptsweets基因可能參與了楊樹(shù)的多個(gè)生物學(xué)過(guò)程,在莖中高表達(dá)的基因可能參與了木質(zhì)部發(fā)育過(guò)程中糖的轉(zhuǎn)運(yùn)。4.為研究ptsweet5、ptsweet7和ptsweet17a的功能,本研究分別創(chuàng)制了ptsweet5、ptsweet7和ptsweet17a的超表達(dá)轉(zhuǎn)基因楊樹(shù)。超表達(dá)ptsweet5促進(jìn)了轉(zhuǎn)基因楊樹(shù)木質(zhì)部和韌皮部的發(fā)育,加速了葉片和根的衰老,并抑制了楊樹(shù)生長(zhǎng)。pi/fda雙熒光染色發(fā)現(xiàn),ptsweet5超表達(dá)株系根細(xì)胞的活力降低。與此相反,超表達(dá)ptsweet7和ptsweet17a均促進(jìn)了楊樹(shù)生長(zhǎng)和木質(zhì)部發(fā)育,說(shuō)明兩個(gè)基因在楊樹(shù)次生生長(zhǎng)中均發(fā)揮重要作用。5.亞細(xì)胞定位和qrt-pcr分析表明,ptsut4基因編碼液泡膜定位的蔗糖轉(zhuǎn)運(yùn)蛋白,并在各組織中表達(dá),在成熟葉、莖、韌皮部和花中具有較高表達(dá)。ptsut4超表達(dá)轉(zhuǎn)基因株系(s1和s12)的株高分別增加了22%和17%,地徑分別增加了9%和7%。另外,2個(gè)超表達(dá)株系第七節(jié)間木質(zhì)部寬度與對(duì)照株系相比,分別增加了32%和21%。超表達(dá)株系的凈光合速率顯著高于對(duì)照,分別高出24%和21%。2個(gè)株系的光合作用增強(qiáng),說(shuō)明ptsut4可能通過(guò)促進(jìn)蔗糖在源端的裝載和在庫(kù)端的卸載而對(duì)光合作用產(chǎn)生正反饋效應(yīng)。光合作用的增強(qiáng)和莖中蔗糖卸載效率的增加,促進(jìn)了楊樹(shù)的高生長(zhǎng)和徑向生長(zhǎng)(次生木質(zhì)部發(fā)育)。本研究分析了27個(gè)ptsweet基因的進(jìn)化和部分糖轉(zhuǎn)運(yùn)特征,其多樣的表達(dá)模式、不同的亞細(xì)胞定位和糖轉(zhuǎn)運(yùn)活性暗示其可能參與木本植物的多個(gè)發(fā)育過(guò)程。進(jìn)一步通過(guò)轉(zhuǎn)基因技術(shù)發(fā)現(xiàn)了ptsweet5、ptsweet7、ptsweet17a和ptsut4在次生生長(zhǎng)中發(fā)揮重要作用。這些結(jié)果為深入研究楊樹(shù)糖轉(zhuǎn)運(yùn)體的功能和調(diào)控提供了線(xiàn)索,并為林木速生或材性改良提供重要的理論和技術(shù)基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國(guó)林業(yè)科學(xué)研究院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S792.11
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本文編號(hào)：2056623