文献类型：期刊文献

中文题名：杨树蔗糖转运体基因PagSUT4的鉴定及功能分析

英文题名：Identification and Functional Analysis of the Populus Sucrose Transporter Gene PagSUT4

作者：张利[1] 徐向东[1] 王丽娟[1] 卢孟柱[1]

第一作者：张利

通信作者：Lu, Mengzhu

机构：[1]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

年份：2016

卷号：52

期号：8

起止页码：21-28

中文期刊名：林业科学

外文期刊名：Scientia Silvae Sinicae

收录：CSTPCD;;EI(收录号：20163902850792);Scopus(收录号：2-s2.0-84988476720);北大核心:【北大核心2014】；CSCD:【CSCD2015_2016】；

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)

语种：中文

中文关键词：杨树;超表达;PagSUT4;糖运输;光合作用;木质部发育

外文关键词：Populus; overexpression; PagSUT4; sugar transport; photosynthesis; xylem development

分类号：S718.46

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)。
[Objective]In higher plants,sugars function as the main energy reserves,important signaling molecules,osmotic adjustment substances and the building blocks for other organic compounds,playing important roles during the entire life cycle.As sucrose transporters（SUTs） in plant,SUTs participate in sucrose loading from source tissues to phloem,transporting in phloem,and unloading from phloem to sink tissues.Therefore,SUTs are important in plant growth and development.There are big differences in sink tissues between woody plants and annual herbs and the carbon allocation mechanism in woody plants needs further investigation.In the present study,to further investigate the roles of Pag SUT4（Gen Bank No.KX545405） in sugar transportation,photosynthesis,and secondary growth,we generated Pag SUT4 overexpression Populus lines and analyzed their phenotypes.[Method]The coding sequence of Pag SUT4 was cloned using gene-specific primers from hybrid poplar clone 84K（Populus alba × P.glandulosa）.The Pag SUT4 expressions in roots,young leaves,mature leaves,primary stem,secondary stem,male flowers,female flowers,xylem and phloem were examined using qRT-PCR.To analyze the subcellular localization of Pag SUT4,the gene was transiently expressed in tobacco leaf epidermal cells fused with YFP to its C-terminus.The coding sequence was cloned into PMDC32 toproduce the35 S∶ Pag SUT4 construct,and the latter was transformed into poplar 84 K mediated by Agrobacterium tumefaciens and the transgenic lines with high expression of Pag SUT4 were selected for further phenotypic analysis.The stem length,ground diameter,photosynthetic rate,intercellular CO2 concentration,conductance to H2 O and transpiration rate of 2-monthold transgenic lines and non-transgenic controls in the greenhouse were measured.Moreover,the anatomy of stem sections of the 7thinternodes from them was performed.[Result]The results showed that Pag SUT4 gene encoding a tonoplast-localized sucrose transporter.The Pag SUT4 gene were ubiquitously expressed and showed high expression level in mature leaves,lignified stem,phloem and flowers.13 transgenic lines with overexpression of Pag SUT4 were obtained and the level of expression in leaves was significantly higher than that in the control.Stomatal conductance,transpiration rate and water use efficiency of the selected transgenic lines（S1 and S12） were higher than that of the non-transgenic controls,while the intercellular CO2 concentration was lower in transgenic lines than that of the controls.The 2 transgenic lines exhibited 24%and 21% higher photosynthetic rate compared with the non-transgenic controls,respectively.The transgenic lines showed22% and 17% increase in stem height,9% and 7% increase in ground diameter,respectively.In addition,the two transgenic lines showed 32% and 21% increase in the xylem width of the seventh internodes,respectively.[Conclusion]Overall,Pag SUT4 functions in mature leaves,secondary stem,phloem and flowers.Overexpression of Pag SUT4 may promote the sucrose efflux from the leaves,the sucrose transport and unloading in the stem,have positive feedback effect on the photosynthesis through the enhancement of sucrose loading in source tissues and the unloading in sink tissues.The enhanced photosynthesis and increased sucrose unloading efficiency in the stem have promoted both height and diameter growth（development of secondary xylem） of the transgenic poplar trees.