木薯MeCML24与MeSAUR1互作调控MeAGPS1a表达的功能验证  

Functional Validation of Interaction Between Cassava MeCML24 and MeSAUR1 to Regulate the Expression of MeAGPS1a

作　　者：郑婉茹 丁凯旋 陆小花 李琳琳 王超群 陈银华[1,2] 姚远 陈新[3] 耿梦婷[1,2] ZHENG Wanru;DING Kaixuan;LU Xiaohua;LI Linlin;WANG Chaoqun;CHEN Yinhua;YAO Yuan;CHEN Xin;GENG Mengting(College of Tropical Crops,Hainan University,Haikou,Hainan 570228,China;Hainan Key Laboratory for Sustainable Utiliza-tion of Tropical Bioresources,Haikou,Hainan 570228,China;Institute of Tropical Biotechnology,Chinese Academy of Tropical Agricultural Sciences,Haikou,Hainan 571101,China;Hainan Seed Station,Haikou,Hainan 571100,China)

机构地区：[1]海南大学热带作物学院,海南海口570228 [2]海南省热带生物资源可持续利用国家重点实验室培育基地,海南海口570228 [3]中国热带农业科学院热带生物技术研究所,海南海口571101 [4]海南省种子总站,海南海口571100

出　　处：《热带作物学报》2023年第1期1-8,共8页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(No.31960058);海南省研究生创新科研课题(No.Hys2020-140)。

摘　　要：木薯是华南地区重要的经济作物,其块根富含淀粉。解析木薯块根淀粉合成调控机理将有助于木薯高产、高淀粉分子改良。AGPase由大亚基和小亚基构成,催化1-磷酸葡萄糖和ATP形成腺苷二磷酸葡萄糖和焦磷酸,其中腺苷二磷酸葡萄糖为淀粉生物合成的底物。AGPase酶是植物淀粉合成的限速酶,提高AGPase酶活性,有利于作物淀粉的积累及产量的提高。木薯MeAGPS1a基因编码的小亚基是AGPase的催化中心,前期研究表明生长素响应因子MeSAUR1作为转录因子正调控MeAGPS1a基因表达,酵母双杂交筛选木薯cDNA文库显示钙调素类似蛋白(CaM-like,CML)成员MeCML24为MeSAUR1的候选互作蛋白。为了确定MeCML24和MeSAUR1的互作关系,本研究从‘SC8’木薯品种基因组克隆了MeCML24基因,该基因的CDS区长度为492 bp,编码163个氨基酸。MeCML24蛋白的理化性质及二级结构分析显示,该蛋白理论等电点pI值4.38,属于亲水性蛋白,α-螺旋占52.76%,无规则卷曲占30.06%,β-转角结构占11.04%。构建酵母双杂交载体BD-MeCML24,自激活实验表明MeCML24不具有自激活活性。酵母双杂交点对点实验发现,共转AD-MeSAUR1和BD-MeCML24质粒的酵母菌株在SD/TLHA+x-α-gal培养基上变蓝,表明MeCML24与MeSAUR1存在互作关系。采用双分子荧光互补(BiFC)实验,将融合蛋白MeSAUR1-nEYFP和MeCML24-cEYFP在烟草叶片中共表达,在激光共聚焦显微镜下检测出黄色荧光蛋白EYFP的荧光信号,进一步证明了MeCML24与MeSAUR1互作。最后利用双荧光素酶实验证明MeCML24与MeSAUR1互作负调控MeAGPS1a基因表达。本研究揭示了木薯MeSAUR1与MeCML24协同调控MeAGPS1a基因表达的机制,发现钙调素类似蛋白MeCML24参与调控木薯块根淀粉合成关键基因MeAGPS1a表达,为利用分子生物学技术培育木薯优良品种提供了理论基础。Cassava is an important cash crop in Southern China,which is enriched with starch in the tuber roots.The analysis of the regulation mechanism of starch synthesis in cassava root would contribute to the improvement of yield and high starch molecular in cassava.AGPase is composed of large subunit and small subunit,and it catalyzes G-1-P and ATP to form ADPG and PPi,in which ADPG is the substrate of starch biosynthesis.Therefore,AGPase is a rate-limiting enzyme in plant starch synthesis,and improving the activity of AGPase is beneficial for the accumulation of crop starch and the improvement of yield.The small subunit encoded by MeAGPS1a is the catalytic center of AGPase in cassava.Previous studies have shown that the growth response factor MeSAUR1 as a transcription factor positively regulates the expression of MeAGPS1a gene,and yeast two-hybrid screening of a cassava cDNA library revealed that the calmodulin-like(CML)member MeCML24 is a candidate interacting protein of MeSAUR1.To determine the interaction between MeCML24 and MeSAUR1,and MeCML24 was cloned from the genome of SC8 cassava variety in this study.The length of CDS region of MeCML24 was 492 bp,encoding 163 amino acids.The physicochemical properties and secondary structure analysis of MeCML24 showed that the value of theoretical pI was 4.38,which belonging to hydrophilic protein withα-helix accounting for 52.76%,random coil accounting for 30.06%,and beta turn accounting for 11.04%.Yeast two-hybrid vector BD-MeCML24 was constructed,and the self-activation experiment showed that MeCML24 had no self-activation.Yeast two-hybrid point-to-point experiment revealed that yeast strains co-transfected with AD-MeSAUR1 and BD-MeCML24 plasmids turned blue on nutrient medium with SD/TLHA+x-α-gal,indicating that MeCML24 interacted with MeSAUR1.The fusion protein MeSAUR1-nEYFP and MeCML24-cEYFP were co-expressed in tobacco leaves by bimolecular fluorescence complementation(BiFC)experiment,and the fluorescence signal of yellow fluorescent protein EYFP was detected under

关 键 词：木薯 MeCML24蛋白 MeSAUR1蛋白 互作蛋白 MeAGPS1a启动子 转录调控 

分 类 号：Q949.748.5[生物学—植物学]