番茄果实成熟过程中SlSWEET7a的功能分析

doi:10.3864/j.issn.0578-1752.2018.15.011

摘要/Abstract

摘要： 【目的】SWEETs（sugars will eventually be exported transporters）是一种糖转运蛋白，参与植物生物进程，对植物生长发育、响应各种胁迫、宿主-病原体的互作发挥作用。克隆番茄SWEET7a，通过构建SlSWEET7a沉默和过表达载体，研究其在糖的转运过程中的作用，为探索SWEETs在植物果实发育过程中的功能提供理论依据。【方法】以Micro-Tom（Solanum lycopersicum）番茄为试材，利用RT-PCR技术从果实中克隆SWEET7a的cDNA全长842bp，进行生物信息学分析，并利用MEGA6.0构建拟南芥进化树，与SlSWEET7a进行蛋白序列同源性分析；利用实时荧光定量PCR技术探明其在果实发育时期的时空表达特征分析，并构建基因的沉默和过表达载体，通过农杆菌介导的果实注射法进行瞬时表达检测构建载体的表达效率；然后进行番茄的遗传转化，获得T1代转基因株系，利用实时荧光定量PCR技术检测绿熟期果实SWEET7a的表达，通过高效液相色谱法检测转基因果实和叶片中糖组成与含量的变化。【结果】SlSWEET7a蛋白结构是由7个跨膜结构域构成的。同源性比对分析结果显示，SlSWEET7a与拟南芥AtSWEET6和AtSWEET8序列同源性较高，都属于SWEETs家族的CladeⅡ。番茄果实各部位的表达分析显示，SlSWEET7a在绿熟期果柄、果实维管束相对表达量最高，转色期和红熟期相对表达量较低。构建SWEET7a沉默（S7a）及过表达载体（OE7a）在番茄果实的瞬时表达，发现OE7a样品果实中SlSWEET7a的表达量是未注射果实的6倍，其SlSWEET7a表达量明显上调，与对照相比，S7a样品果实中SlSWEET7a明显下调了5倍。在番茄中的遗传转化中卡那霉素抗性筛选获得10株可能的超表达T0代植株，PCR鉴定得到了SlSWEET7a超表达8株；沉默株系经除草剂筛选，获得14株，PCR检测得到10株沉默株系。T1代植株的实时定量分析显示，过表达SlSWEET7a植株发生转基因沉默现象，SlSWEET7a表达量显著低于正常植株，而沉默植株表达量也降低，说明获得的过表达植株也发生了基因沉默。果糖、葡萄糖和蔗糖含量测定结果表明，降低番茄中SlSWEET7a的表达，植株成熟叶片和绿熟期果实中果糖、葡萄糖和蔗糖含量均高于对照，尤其是叶片中蔗糖含量显著高于对照，这说明SlSWEET7a对细胞中蔗糖的易化扩散起着重要作用。【结论】SlSWEET7a对叶片中蔗糖向源组织韧皮部的装载及果实果柄、维管束的运输、卸载起重要调控作用。

关键词: 番茄, SWEET7a, 糖转运, 载体构建, 表达分析

Abstract: 【Objective】 The SWEETs (Sugars Will Eventually Be Exported Transporters) is a kind of sugar transporters which are involved in plant biological processes and play key roles in plant growth and development in response to various stresses and host-pathogen interaction. SWEET7a was cloned and its function was analyzed to provide the theoretical foundation for exploring the function of SWEETs during fruit development in plant by constructing the silence and overexpression of SlSWEET7a.【Method】Using Micro-Tom (Solanum lycopersicum) as a test material, the 842 bp full-length SlSWEET7a cDNA was cloned in fruits. The phylogenetic tree of SWEET proteins from Arabidopsis was constructed by using MEGA6.0, and homology of SlSWEET7a protein sequences was analysed compared with Arabidopsis. RT-PCR was performed to analyse the spatiotemporal expression of SlSweet7a during the fruits development periods. The SlSWEET7a silence vector and overexpression vector were constructed and transformed into tomato by Agrobacterium-mediated genetic transformation. The efficiency of two vectors was examined by the transient expression of Agrobacterium tumefaciens injection method. The expressions of SWEET7a in T1 generation transgenic green fruits were studied by quantitative PCR. The changes of sugar composition and content in transgenic fruits and leaves were detected by high performance liquid chromatography.【Result】The bioinformatics analysis of protein sequence showed that the SlSWEET7a is consisted of seven transmembrane domains. SlSWEET7a belongs to the CladeⅡof the SWEETs gene family, which was highly homologous to AtSWEET6 and AtSWEET8 in Arabidopsis thaliana. The analysis of spatiotemporal expression indicated that SlSWEET7a expression level was the highest at stalks and vascular bundles of mature green stage in tomato fruits, while the relative expression level was lower in breaker fruits and red ripping stage. The transient expression of SWEET7a silencing (S7a) and overexpression (OE7a) vector in tomato fruits was observed. The expression level of SlSWEET7a in fruit of OE7a was 6 times higher than that of non-injected fruit, which was significantly up-regulated compared with control. However, the expression level of SlSWEET7a in S7a was significantly decreased 5 times. Ten overexpression lines were obtained by kanamycin resistance screening, and eight overexpression lines were obtained by PCR. Fourteen silencing lines were screened by phosphinothricin and 10 transgenic silence lines were obtained by PCR. Real-time quantitative PCR analysis of the SWEET7a expression in T1 generation lines revealed that gene silencing occurred in overexpressed plants. The expression level of SlSWEET7a-overexpressing transgenic plants was significantly lower than that in wild plants, so did the silencing plants. Those results explained that the overexpressing plants also had the phenomenon of gene silencing. The determination of contents of fructose, glucose, sucrose showed that the silencing and overexpressing plants were higher than that of the wild type in the leaves and fruits after reducing expression of SlSWEET7a in tomato. Especially, the sucrose content of leaves was significantly increased. This showed that SlSWEET7a plays an important role in the facilitated diffusion of sucrose in cells.【Conclusion】SlSWEET7a plays an important role in regulating the loading of sucrose into the phloem of fruit tissue, the transportation and unloading of fruit stalks and vascular bundles.

Key words: tomato, SWEET7a, sugar transporter, vector construction, expression analysis

程杰，张新圣，李安琪，姜晶. 番茄果实成熟过程中SlSWEET7a的功能分析[J]. 中国农业科学, 2018, 51(15): 2958-2968.

CHENG Jie, ZHANG XinSheng, LI AnQi, JIANG Jing. Functional Analysis of SlSWEET7a Gene During Maturation of Tomato Fruits[J]. Scientia Agricultura Sinica, 2018, 51(15): 2958-2968.

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