本文選題：楊樹 + 超表達(dá)��； 參考：《林業(yè)科學(xué)》2016年08期

【摘要】：【目的】植物蔗糖轉(zhuǎn)運(yùn)體SUTs參與蔗糖由源組織到韌皮部的裝載、韌皮部的運(yùn)輸和由韌皮部到庫組織的卸載過程,對(duì)植物生長(zhǎng)發(fā)育至關(guān)重要。本研究通過在楊樹中超表達(dá)Pag SUT4基因(Gen Bank登錄號(hào):KX545405)并分析轉(zhuǎn)基因株系的表型,探討Pag SUT4在楊樹糖轉(zhuǎn)運(yùn)、光合作用和次生生長(zhǎng)中的作用。【方法】利用同源基因克隆技術(shù),克隆銀腺楊Pag SUT4基因;利用實(shí)時(shí)熒光定量PCR技術(shù),分析銀腺楊Pag SUT4基因在根、幼葉、成熟葉、初生莖、次生莖、雄花和雌花及木質(zhì)部、韌皮部中的表達(dá)。通過在煙草葉片瞬時(shí)轉(zhuǎn)化35 S∶YFP-Pag SUT4,對(duì)Pag SUT4的亞細(xì)胞定位進(jìn)行分析。利用Gateway技術(shù),將Pag SUT4編碼區(qū)序列重組進(jìn)入PMDC32載體,從而構(gòu)建35 S∶Pag SUT4載體。利用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化銀腺楊,并選取Pag SUT4表達(dá)量較高的2個(gè)轉(zhuǎn)基因株系用于表型分析。對(duì)在溫室生長(zhǎng)2個(gè)月的轉(zhuǎn)基因株系和對(duì)照植株的株高、地徑、凈光合速率、胞間CO2濃度、氣孔導(dǎo)度和蒸騰速率進(jìn)行測(cè)定。此外,通過組織切片對(duì)轉(zhuǎn)基因和對(duì)照植株的第7節(jié)間的解剖學(xué)特征進(jìn)行分析�！窘Y(jié)果】Pag SUT4基因編碼的蔗糖轉(zhuǎn)運(yùn)蛋白定位于液泡膜。Pag SUT4基因在各組織中均有表達(dá),并在成熟葉、次生莖、韌皮部和花中具有較高表達(dá)。基于實(shí)時(shí)熒光定量PCR分析,確認(rèn)獲得13個(gè)Pag SUT4超表達(dá)株系,其Pag SUT4在葉片中的表達(dá)量均顯著高于對(duì)照。2個(gè)Pag SUT4超表達(dá)株系(S1和S12)的氣孔導(dǎo)度、蒸騰速率和水分利用效率均高于對(duì)照,而其胞間CO2濃度低于對(duì)照;凈光合速率顯著高于對(duì)照,分別高出24%和21%;與對(duì)照相比,株高分別增加22%和17%,地徑分別增加9%和7%。莖解剖分析發(fā)現(xiàn),2個(gè)Pag SUT4超表達(dá)株系第7節(jié)間木質(zhì)部寬度與對(duì)照株系相比分別增加32%和21%�！窘Y(jié)論】Pag SUT4基因主要在成熟葉、次生莖和韌皮部及花中發(fā)揮作用。楊樹超表達(dá)Pag SUT4可能通過促進(jìn)葉片中糖的外運(yùn)和莖中糖的運(yùn)輸與卸載,提高蔗糖在源端的裝載及在庫端的卸載效率進(jìn)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)楊樹的高生長(zhǎng)和徑向生長(zhǎng)(次生木質(zhì)部發(fā)育)。
[Abstract]:[objective] the plant sucrose transporter SUTs is involved in the loading of sucrose from source tissue to phloem, the transport of sucrose from phloem to reservoir, and the unloading process from phloem to reservoir, which is very important for plant growth and development. The purpose of this study was to investigate the role of Pag SUT4 in sugar transport, photosynthesis and secondary growth of poplar by overexpression of Pag SUT4 gene in poplar (Pag SUT4 Bank accession number: KX545405) and analysis of phenotype of transgenic lines. [methods] homologous gene cloning technique was used to investigate the role of Pag SUT4 in poplar sugar transport, photosynthesis and secondary growth. Pag SUT4 gene was cloned and the expression of Pag SUT4 gene in root, young leaf, mature leaf, primary stem, secondary stem, male flower and female flower, xylem and phloem were analyzed by real-time fluorescence quantitative PCR. The subcellular localization of Pag SUT4 was analyzed by transient transformation of 35 S:YFP-Pag SUT4 in tobacco leaves. Using Gateway technology, Pag SUT4 coding region sequence was recombined into PMDC32 vector, and then 35 S:Pag SUT4 vector was constructed. Agrobacterium tumefaciens-mediated transformation of silver poplar was carried out, and two transgenic lines with high Pag SUT4 expression were selected for phenotypic analysis. The plant height, ground diameter, net photosynthetic rate, intercellular CO2 concentration, stomatal conductance and transpiration rate of transgenic lines and control plants grown in greenhouse for 2 months were measured. In addition, the anatomical characteristics of the seventh internode of transgenic and control plants were analyzed by tissue sections. [results] the sucrose transporter encoded by Pag SUT4 gene was located in the vacuolar membrane. Pag SUT4 gene was expressed in all tissues and was expressed in mature leaves. The secondary stem, phloem and flower have high expression. Based on real-time fluorescence quantitative PCR analysis, 13 Pag SUT4 overexpression lines were obtained, and their Pag SUT4 expression in leaves was significantly higher than that in control. The stomatal conductance of 2 Pag SUT4 overexpression lines S12 and S1) was significantly higher than that of control. The transpiration rate and water use efficiency were higher than the control, but the intercellular CO2 concentration was lower than that of the control, the net photosynthetic rate was significantly higher than that of the control, which was 24% and 21% higher than that of the control, and the plant height increased 22% and 17%, and the ground diameter increased 9% and 7%, respectively, compared with the control. Stem anatomical analysis showed that the width of the seventh internode xylem of two Pag SUT4 superexpressed lines was increased by 32% and 21%, respectively. [conclusion] Pag SUT4 gene plays a major role in mature leaves, secondary stems and phloem and flowers. The overexpression of Pag SUT4 in poplar may increase the loading efficiency of sucrose at the source end and the unloading efficiency of sucrose at the storage end by promoting the transport of sugar in leaves and the transport and unloading of sugar in stem, thus producing a positive feedback effect on photosynthesis. The increase of photosynthesis and the increase of sucrose unloading efficiency in stem promoted the high growth and radial growth of poplar (secondary xylem development).
【作者單位】： 國(guó)家林業(yè)局林木培育重點(diǎn)實(shí)驗(yàn)室中國(guó)林業(yè)科學(xué)研究院林業(yè)研究所;
【基金】：“十二五”863計(jì)劃課題“林木優(yōu)質(zhì)、速生性狀調(diào)控基因的分離及育種技術(shù)研究”(2013AA102702)
【分類號(hào)】：S792.11
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本文編號(hào)：1815019