红肉火龙果糖转运蛋白HpSWEET4b基因克隆、表达和转运活性鉴定  

Cloning,expression analysis and transport activity assay of sugar transporter gene HpSWEET4b in red pitaya(Hylocereus polyrhizus)

作　　者：郑乾明 晏霜 解璞[1] 王红林 ZHENG Qian-ming;YAN Shuang;XIE Pu;WANG Hong-lin(Guizhou Institute of Pomology Science,Guizhou Academy of Agricultural Sciences,Guiyang 550006,China;Ministry of Agriculture and Rural Affairs Key Laboratory of Crop Genetic Resources and Germplasm Innovation in Karst Region,Ministry of Agriculture and Rural Affairs,Guizhou Academy of Agricultural Sciences,Guiyang 550006,China)

机构地区：[1]贵州省农业科学院贵州省果树科学研究所,贵阳550006 [2]贵州省农业科学院农业农村部喀斯特山区作物基因资源与种质创新重点实验室,贵阳550006

出　　处：《西南农业学报》2024年第12期2739-2748,共10页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(32060674);贵州省农业科学院国家自然科学基金后补助项目(黔农科院国基后补助[2021]61);贵州省高层次创新型人才项目“百层次人才”(黔科合平台人才-GCC[2022]025-1);贵州省科技计划项目(黔科合中引地[2023]033)。

摘　　要：【目的】探明红肉火龙果(Hylocereus polyrhizus)糖转运蛋白SWEET HpSWEET4b基因的表达模式、亚细胞定位和糖转运活性,为调控果实可溶性糖积累提供依据。【方法】采用反转录PCR(RT-PCR)从果肉cDNA克隆HpSWEET4b基因,开展序列比对和系统进化分析。采用实时定量PCR(qRT-PCR)研究基因表达,融合荧光蛋白在烟草叶肉细胞瞬时表达研究亚细胞定位。在酵母己糖吸收缺陷株系表达HpSWEET4b,采用酵母生长互补和胞外酸化方法研究糖转运活性。【结果】HpSWEET4b基因的开放阅读框(ORF)长度为744 bp,编码247个氨基酸残基,蛋白相对分子质量为27.34 kDa,理论等电点为9.41。HpSWEET4b蛋白含有2个“MtN3_slv”(PFAM编号:PF03083.19)结构域,以及7个跨膜结构域。系统进化分析表明,HpSWEET4b与拟南芥AtSWEET4~AtSWEET8聚为一类,属于SWEET家族II类。HpSWEET4b在茎的表达量极低,在授粉后20 d的果肉表达量最高,并随果实发育显著下降。融合荧光蛋白在烟草叶肉细胞瞬时表达证明HpSWEET4b主要定位于细胞膜。经酵母生长互补检测,HpSWEET4b具有葡萄糖、果糖、2-脱氧葡萄糖和甘露糖转运活性,不具有山梨醇、甘露醇和半乳糖转运活性。葡萄糖和果糖均促进酵母胞外酸化,进一步证明HpSWEET4b的葡萄糖和果糖转运活性。【结论】红肉火龙果HpSWEET4b属于SWEET家族II类成员,定位于细胞膜,具有葡萄糖和果糖等己糖转运活性,主要在授粉后20~23 d的果肉参与质外体和胞质之间的己糖跨膜转运。[Objective]The expression pattern,subcellular localization and sugar transport activity of(Hylocereus polyrhizus)SWEET Hp-SWEET4b gene are investigated to provide the scientific basis for regulating soluble sugar accumulation in red pitaya.[Method]The sequence alignment and phylogenetic relationship of HpSWEET4b gene cloned by RT-PCR from fruit pulp cDNA was analyzed.The expression patern and subcellular localization of HpSWEET4b gene were analyzed by Real-time quantitative PCR(qRT-PCR)and transient expression of fusion fluorescent protein in tobacco mesophyll cells respectively.The sugar transport activity of HpSWEET4b gene over-expressed in the hexose up-take-defected yeast strain was assayed by yeast growth complementation and extracellular acidification method.[Result]The ORF length of HpSWEET4b gene was 744 bp with encoded 247 amino acid residues.The protein relative molecular mass and theoretical isoelectric point of HpSWEET4b gene were 27.34 kDa and 9.41respectively.HpSWEET4b protein contained two‘MtN3_slv'(PF03083.19)structural do-mains and seven transmembrane domains.Phylogenetic analysis showed that HpSWEET4b and Arabidopsis AtSWEET4-AtSWEET8 were clus-tered into the same group belonging to class II of SWEET family.The expression level of HpSWEET4b gene was very low in stem.The expres-sion level of HpSWEET4b gene was the highest in fruit pulp on 2Od after pollination and significantly decreaseed with fruit development.The subcellular localization assay demonstrateed that HpSWEET4b gene was mainly localized on plasma membrane according to expression of fu-sion fluorescent protein in tobacco mesophyll cells.HpSWEET4b gene had the transport activity to glucose,fructose,2-deoxyglucose and man-nose,and no transport activity to sorbitol,mannitol and galactose by yeast growth complementation assay.Glucose and fructose both could pro-mote yeast extracellular acidification,which further indicated that HpSWEET4b gene had the transport activity of glucose and fructose.[Con-clusion]HpSWEET4b gene of red pitaya be

关 键 词：红肉火龙果 果实发育 糖转运蛋白SWEET 基因克隆 表达分析 转运活性鉴定 

分 类 号：S667[农业科学—果树学]