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

Abstract

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

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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Functional Analysis of SlSWEET7a Gene During Maturation of Tomato Fruits

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