甘薯蔗糖转运蛋白的功能分析

Functional analysis on sucrose transporters in sweet potato

摘要:

甘薯（Ipomoea batatas）是重要的粮食和工业加工原料作物。蔗糖是植物体内碳水化合物长距离转运的主要形式，蔗糖转运蛋白（sucrose transporter，SUT）在植物的生长代谢中调控蔗糖的跨膜运输和分配，在韧皮部介导的源-库蔗糖运输和为库组织供应蔗糖的生理活动中起关键作用。本研究根据不同淀粉性状甘薯块根中差异表达的2个SUT基因转录本，进行cDNA末端快速扩增（rapid amplification of cDNA ends，RACE）克隆，获得IbSUT₆₂₇₈₈和IbSUT₈₁₆₁₆的全长cDNA序列；通过系统发育分析明确其分类；通过在本氏烟草（Nicotiana benthamiana）中瞬时表达明确其亚细胞定位；通过酵母功能互补系统鉴定IbSUT₆₂₇₈₈和IbSUT₈₁₆₁₆是否具有吸收、转运蔗糖和己糖的能力。通过实时荧光定量PCR （real-time fluorescence quantitative polymerase chain reaction，RT-qPCR）分析IbSU₆₂₇₈₈和IbSUT₈₁₆₁₆在甘薯各器官中的表达特征；通过蘸花法得到外源表达IbSUT₆₂₇₈₈和IbSUT₈₁₆₁₆基因的拟南芥（Arabidopsis thaliana）植株，比较与野生型拟南芥的淀粉和糖含量的差异。结果表明，IbSUT₆₂₇₈₈和IbSUT₈₁₆₁₆分别编码505个和521个氨基酸的SUT蛋白，均属于SUT1亚家族。IbSUT₆₂₇₈₈和IbSUT₈₁₆₁₆均定位于细胞膜，在酵母系统中具有转运蔗糖、葡萄糖和果糖的能力。此外，IbSUT₆₂₇₈₈还具有转运甘露糖的能力。IbSUT₆₂₇₈₈在甘薯叶片、侧枝和茎中的表达量更高，IbSUT₈₁₆₁₆在侧枝、茎和块根中表达量更高。IbSUT₆₂₇₈₈和IbSUT₈₁₆₁₆在拟南芥中异源表达后，植株可以正常生长，但生物量增加。IbSUT₆₂₇₈₈的异源表达增加了拟南芥植株叶片可溶性糖含量、叶片大小和种子千粒重；IbSUT₈₁₆₁₆的异源表达增加了拟南芥植株叶片、根尖的淀粉积累量和种子千粒重，但减少了可溶性糖含量。本研究结果表明，IbSUT₆₂₇₈₈和IbSUT₈₁₆₁₆可能是调控甘薯蔗糖和糖含量性状的重要基因，在细胞膜上进行着蔗糖的跨膜运输、蔗糖进出库组织、韧皮部蔗糖的运输与卸载等生理功能，在拟南芥中异源表达造成的性状改变说明其在提高其他植物或作物产量中的应用潜力。本研究为揭示甘薯淀粉和糖代谢及重要品质性状形成机制提供了重要信息。

Abstract:

Sweet potato is an important food crop that can also be used as an industrial raw material. Sucrose is the main form of long-distance carbohydrate transport in plants, and sucrose transporter (SUT) regulates the transmembrane transport and distribution of sucrose during plant growth and metabolism. Moreover, SUT plays a key role in phloem mediated source-to-sink sucrose transport and physiological activities, supplying sucrose for the sink tissues. In this study, the full-length cDNA sequences of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were obtained by rapid amplification of cDNA ends (RACE) cloning according to the transcripts of the two SUT coding genes which were differentially expressed in sweet potato storage roots with different starch properties. Phylogenetic analysis was performed to clarify the classification of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆. The subcellular localization of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ was determined by transient expression in Nicotiana benthamiana. The function of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in sucrose and hexose absorption and transport was identified using yeast functional complementarity system. The expression pattern of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in sweet potato organs were analyzed by real-time fluorescence quantitative PCR (RT-qPCR). Arabidopsis plants heterologous expressing IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ genes were obtained using floral dip method. The differences in starch and sugar contents between transgenic and wild-type Arabidopsis were compared. The results showed IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ encoded SUT proteins with a length of 505 and 521 amino acids, respectively, and both proteins belonged to the SUT1 subfamily. IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were located in the cell membrane and were able to transport sucrose, glucose and fructose in the yeast system. In addition, IbSUT₆₂₇₈₈ was also able to transport mannose. The expression of IbSUT₆₂₇₈₈ was higher in leaves, lateral branches and main stems, and the expression of IbSUT₈₁₆₁₆ was higher in lateral branches, stems and storage roots. After IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were heterologously expressed in Arabidopsis, the plants grew normally, but the biomass increased. The heterologous expression of IbSUT₆₂₇₈₈ increased the soluble sugar content, leaf size and 1 000-seed weight of Arabidopsis plants. Heterologous expression of IbSUT₈₁₆₁₆ increased starch accumulation in leaves and root tips and 1 000-seed weight of seeds, but decreased soluble sugar content. The results obtained in this study showed that IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ might be important genes regulating sucrose and sugar content traits in sweet potato. They might carry out physiological functions on cell membrane, such as transmembrane transport of sucrose, sucrose into and out of sink tissue, as well as transport and unloading of sucrose into phloem. The changes in traits result from their heterologous expression in Arabidopsis indicates their potential in improving the yield of other plants or crops. The results obtained in this study provide important information for revealing the functions of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in starch and glucose metabolism and formation mechanism of important quality traits in sweet potato.