马铃薯淀粉酶StBAM9互作蛋白的鉴定及其互作机制分析  

Identification of potato amylase StBAM9 interacting protein and analysis of the interaction mechanism

作　　者：杜鹃[1] 彭晓君[1,2] 侯娟 刘腾飞[1,4] 刘增 宋波涛[1] DU Juan;PENG Xiao-Jun;HOU Juan;LIU Teng-Fei;LIU Zeng;SONG Bo-Tao(National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops/Key Laboratory of Potato Biology and Biotechnology,Ministry of Agriculture and Rural Affairs/Huazhong Agricultural University,Wuhan 430070,Hubei,China;People’s Government of Baren Township,Aktau County,Kizilsu Kirgiz Autonomous Prefecture 845555,Xinjiang,China;College of Horticulture,Henan Agricultural University,Zhengzhou 450002,Henan,China;College of Food Science and Engineering,Shandong Agricultural University,Tai’an 271018,Shandong,China)

机构地区：[1]果蔬园艺作物种质创新与利用全国重点实验室/农业农村部马铃薯生物学与生物技术重点实验室/华中农业大学,湖北武汉430070 [2]阿克陶县巴仁乡人民政府,新疆克孜勒苏柯尔克孜自治州845555 [3]河南农业大学园艺学院,河南郑州450002 [4]山东农业大学食品科学与工程学院,山东泰安271018

出　　处：《作物学报》2023年第10期2643-2653,共11页Acta Agronomica Sinica

基　　金：贵州省科技支撑计划(农业领域重点项目)生物育种先导性项目(黔科合支撑[2022]重点030-3);国家自然科学基金项目(31671749)资助。

摘　　要：本实验室前期研究表明β-淀粉酶9(StBAM9)在马铃薯抗低温糖化中具有重要作用,但其并无β-淀粉酶活性。为了研究StBAM9在马铃薯抗低温糖化中的功能机制,我们构建了低温贮藏后块茎cDNA酵母双杂交文库,并以StBAM9蛋白为诱饵,对其进行了互作蛋白的筛选和分析,结果显示分别以全长StBAM9和截去转运肽的StBAM9为诱饵筛选到的候选互作蛋白中有12个是共有的。酵母双杂交结果显示有4个蛋白(StDUF842、StTPR01660、StTPR22129和StTPR45174)与StBAM9互作。进一步通过谷胱甘肽-S-转移酶融合蛋白沉降技术验证表明,其中的2个蛋白StTPR01660和StTPR4517与StBAM9互作。双分子荧光互补结果显示只有StTPR01660与StBAM9互作于淀粉粒上,而StTPR01660自身定位于细胞质。因此,我们推测StBAM9可能通过从细胞质中招募StTPR01660到淀粉粒上发挥淀粉降解的功能。Previous research conducted in our laboratory has demonstrated the crucial role of StBAM9(β-Amylase 9)in the resis-tance of potatoes to cold-induced sweetening(CIS),although StBAM9 lacksβ-Amylase activity.To investigate the mechanism,we generated a yeast two-hybrid library of tuber cDNA following low-temperature storage and screened for potential interacting proteins with StBAM9 as bait.The results revealed that 12 of the identified interacting proteins were common among both full-length and truncated transport peptide StBAM9 bait screens.Among them,four proteins(StDUF842,StTPR01660,StTPR22129,and StTPR45174)had significant interactions with StBAM9 in yeast two-hybrid assay.Subsequently,two of these proteins,StTPR01660 and StTPR4517,were identified as the interactors with StBAM9 through Glutathione-S-transferase(GST)pull-down experiments.Bimolecular fluorescence complementation(BiFC)assays demonstrated that only StTPR01660 was co-localized with StBAM9 on starch granules,while StTPR01660 itself was observed in the cytoplasm.In conclusion,StBAM9 may recruit StTPR01660 from the cytoplasm to starch granules,potentially enabling starch degradation.

关 键 词：马铃薯 低温糖化 淀粉降解 Β-淀粉酶 蛋白互作 

分 类 号：S532[农业科学—作物学]