拟南芥HHP2基因对韧皮部蔗糖装载的调控功能  

Function of Arabidopsis HHP2 gene in regulating sucrose phloem loading

作　　者：于修远 徐启玉 尹世娇 陈少林[1,2] LIESCHE Johannes YU Xiuyuan;XU Qiyu;YIN Shijiao;CHEN Shaolin;LIESCHE Johannes(College of Life Sciences,Northwest A&F niversity,Yangling,Shaanxi 712100,China;Biomass Energy Center for Arid and Semiarid Lands,Northwest A&F University,Yangling,Shaanxi 712100,China;State Key Laboratory of Stress Biology for Arid Areas,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]西北农林科技大学旱区生物质能研究中心,陕西杨凌712100 [3]西北农林科技大学旱区作物逆境生物学国家重点实验室,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2024年第8期122-132,142,共12页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(K3050218117)。

摘　　要：【目的】探究膜蛋白HHP2在韧皮部蔗糖装载中的作用与机制,为作物增产增效提供理论依据。【方法】利用多种生物信息学分析方法,对HHP2及其同源蛋白序列以及HHP2基因的转录表达谱进行分析,以预测其功能并发掘其他同源蛋白。使用荧光共聚焦显微镜对HHP2蛋白和蔗糖转运蛋白SUC2进行共定位分析,利用荧光共振能量转移试验检测HHP2与SUC2蛋白的互作,通过七叶树苷摄取试验检测HHP2对SUC2蛋白的直接影响。通过实时荧光定量PCR和Western-Blot试验,分析和比较hhp2突变体SUC2基因的转录水平和蛋白水平;利用离子色谱方法分析和比较hhp2突变体的韧皮部蔗糖输出效率;采用AlphaFold和Autodock软件,对HHP2蛋白结构及HHP2和蔗糖分子之间的潜在对接能力进行分析。【结果】HHP2同源蛋白共有5个保守模体和1个保守结构域。HHP2基因在拟南芥叶和茎等蔗糖输出器官中低表达,在根、花和种子等蔗糖需求器官中高表达。在本氏烟草远轴面表皮细胞中瞬时表达HHP2,发现其与SUC2蛋白共定位。HHP2与SUC2蛋白存在互作,且HHP2能抑制SUC2的蔗糖转运活性。HHP2缺失突变分析表明,HHP2是SUC2的负调控因子,HHP2缺失突变导致SUC2转录水平提高至6倍左右,蛋白水平提高至1.3倍左右,hhp2缺失突变体的蔗糖转运效率提高至2倍左右。分子对接分析表明,HHP2可能通过与蔗糖结合发挥其调控功能。【结论】HHP2是SUC2的负调控因子,参与调控植物体内蔗糖的转运与分配,可能是分子育种的潜在靶标。【Objective】This study explored the role of HHP2 in regulating sucrose phloem loading in Arabidopsis thaliana to provide basis for increasing plant yield and growth performance.【Method】A variety of bioinformatics analysis tools were used to identify conserved sequences,homologous genes and expression profile of HHP2,so as to infer its function and discover other homologous proteins.The colocalization analysis of HHP2 protein and sucrose transporter SUC2 was carried out by fluorescence confocal microscopy.The protein interaction between HHP2 and SUC2 was detected by fluorescence resonance energy transfer experiment.The direct effect of HHP2 on SUC2 protein was detected by esculin uptake experiment.Relative quantification of SUC2 gene expression and protein levels was performed on hhp2 mutant using real-time quantitative PCR and Western-Blot.The phloem sucrose export efficiency in hhp2 mutant was determined using ion chromatography on phloem exudates.AlphaFold and Autodock software were used to analyze the structure of HHP2 protein and potential docking between HHP2 and sucrose molecules.【Result】Five conserved motifs and 1 conserved domain shared by HHP2 homologous proteins were identified.HHP2 gene was lowly expressed in sucrose-exporting organs such as leaves and stems,while highly expressed in sucrose-requiring organs such as roots,flowers and seeds.HHP2 gene was cloned and transient expressed in the abaxial surface epidermal cells of Nicotiana benthamiana leaves,demonstrating that it co-localized with SUC2 protein.HHP2 protein interacted with SUC2 protein and it inhibited the sucrose transport activity of SUC2 protein.The deletion mutation analysis of HHP2 gene suggested that HHP2 was a negative regulator of SUC2.HHP2 deficiency resulted in increases in SUC2 transcript levels to about 6 times,in protein levels to about 1.3 times,and in sucrose transport efficiency to about 2 times.Molecular docking analysis indicated that HHP2 protein may perform its regulatory function by binding sucrose.【Conclusi

关 键 词：拟南芥 膜蛋白 蔗糖转运 调控因子 

分 类 号：Q943.2[生物学—植物学]