杨树蔗糖转运体基因PagSUT4的鉴定及功能分析

发布时间：2018-04-28 11:08

  本文选题：杨树 + 超表达　； 参考：《林业科学》2016年08期

【摘要】：【目的】植物蔗糖转运体SUTs参与蔗糖由源组织到韧皮部的装载、韧皮部的运输和由韧皮部到库组织的卸载过程,对植物生长发育至关重要。本研究通过在杨树中超表达Pag SUT4基因(Gen Bank登录号:KX545405)并分析转基因株系的表型,探讨Pag SUT4在杨树糖转运、光合作用和次生生长中的作用。【方法】利用同源基因克隆技术,克隆银腺杨Pag SUT4基因;利用实时荧光定量PCR技术,分析银腺杨Pag SUT4基因在根、幼叶、成熟叶、初生茎、次生茎、雄花和雌花及木质部、韧皮部中的表达。通过在烟草叶片瞬时转化35 S∶YFP-Pag SUT4,对Pag SUT4的亚细胞定位进行分析。利用Gateway技术,将Pag SUT4编码区序列重组进入PMDC32载体,从而构建35 S∶Pag SUT4载体。利用农杆菌介导法转化银腺杨,并选取Pag SUT4表达量较高的2个转基因株系用于表型分析。对在温室生长2个月的转基因株系和对照植株的株高、地径、净光合速率、胞间CO2浓度、气孔导度和蒸腾速率进行测定。此外,通过组织切片对转基因和对照植株的第7节间的解剖学特征进行分析。【结果】Pag SUT4基因编码的蔗糖转运蛋白定位于液泡膜。Pag SUT4基因在各组织中均有表达,并在成熟叶、次生茎、韧皮部和花中具有较高表达。基于实时荧光定量PCR分析,确认获得13个Pag SUT4超表达株系,其Pag SUT4在叶片中的表达量均显著高于对照。2个Pag SUT4超表达株系(S1和S12)的气孔导度、蒸腾速率和水分利用效率均高于对照,而其胞间CO2浓度低于对照;净光合速率显著高于对照,分别高出24%和21%;与对照相比,株高分别增加22%和17%,地径分别增加9%和7%。茎解剖分析发现,2个Pag SUT4超表达株系第7节间木质部宽度与对照株系相比分别增加32%和21%。【结论】Pag SUT4基因主要在成熟叶、次生茎和韧皮部及花中发挥作用。杨树超表达Pag SUT4可能通过促进叶片中糖的外运和茎中糖的运输与卸载,提高蔗糖在源端的装载及在库端的卸载效率进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,促进杨树的高生长和径向生长(次生木质部发育)。
[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).
【作者单位】： 国家林业局林木培育重点实验室中国林业科学研究院林业研究所;
【基金】：“十二五”863计划课题“林木优质、速生性状调控基因的分离及育种技术研究”(2013AA102702)
【分类号】：S792.11
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本文编号：1815019