IbCPK28调控甘薯块根形成与发育的分子机制研究

Sweetpotato is an important tuberous root crops for food, industrial material and bio-energy resource. The biological processes of formation and development of the tuberous root are complex, which are regulated by many genes. As far, few genes related to tuberous root formation and development have been reported, and the molecular regulation mechanism of tuberous root formation and development remains unclear. Based on the root RNA-Seq data analysis of a variant sweetpotato line in our previous studies, a calcium-dependent protein kinase 28 (IbCPK28) gene, with the highest expression in the tuberous root than in other tissues, was selected and cloned from differentially expressed genes. IbCPK28 was transferred into sweetpotato obtaining transgenic sweetpotato plants, which were then domesticated and transplanted to the field. Compared with the WT plants, the number of tuberous roots per plant of IbCPK28-overexpressing sweetpotato lines increased by 44%~85%, and the yield per plant increased by 19%~66%. Moreover, dry matter ratio in the tuberous roots of IbCPK28-overexpressing sweetpotato lines were significantly increased. The IbCPK28-RNA interfered sweetpotato lines showed the opposite trend. Therefore, it is speculated that IbCPK28 is related to the formation and development of tuberous root in sweetpotato. Based on these previous findings, in this study, systematic morphological and histological observation of transgenic sweetpotato plants will be performed, and physiological and biochemical indexes of transgenic sweetpotato plants will be measured. Transcriptome analysis of leaves and tuberous roots of transgenic sweetpotato plants will be performed. The upstream genes, interacting proteins and phosphorylation substrate of IbCPK28 will be screened and identified. The function of IbCPK28 will be clarified, and its molecular regulation mechanism of tuberous root formation and development in sweetpotato will be explored. The success of this research will not only provide theoretical basis for the study of gene regulatory network about tuberous root formation and development of sweetpotato, but also provide important gene material for breeding sweetpotato varieties with high yield and good quality.

甘薯是重要的粮食、工业原料和新型能源块根作物，其块根形成发育是一个复杂的生物学过程，受到诸多基因的调控，目前甘薯块根形成与发育的相关基因鲜有报道，且分子调控机制尚不清楚。本课题组通过对创制的变异品系进行根系转录组分析，克隆到差异表达的钙依赖蛋白激酶基因IbCPK28，在块根中表达量最高，其过表达甘薯植株驯化并移栽大田，单株块根数量增加44%~85%，单株产量提高19%~66%，且干物率显著提高，RNAi干扰植株则相反，推测它是一个与甘薯块根形成与发育相关的基因。本项目将在此基础上，对转基因甘薯植株进行系统的形态学与组织学观察，生理生化指标测定，并对其叶片和块根进行转录组分析；对IbCPK28的上游基因、互作蛋白及磷酸化底物进行筛选并鉴定，充分明确其功能，解析其调控甘薯块根形成与发育的分子机理。研究结果为揭示甘薯块根形成与发育的分子机制提供参考，同时为高产优质甘薯新品种的改良提供新基因。

目前甘薯块根形成与发育的相关基因鲜有报道，且分子调控机制尚不清楚。本项目通过对根系转录组分析，筛选出一个块根中高表达的钙依赖蛋白激酶基因IbCPK28，其受到IAA、GA的显著诱导表达，启动子在主根中特异启动。IbCPK28过表达株系块根数显著增多，单株鲜重和单株干重均显著增加，单株干物率显著增加；RNAi干扰株系的块根形成困难，单株块根数量较野生型显著减少，单株干鲜重显著降低。IbbZIP11能够靶标IbCPK28的启动子区域抑制其转录。IbbZIP11基因负调控甘薯块根发育，过表达株系块根形成困难，不结薯；RNAi干扰株系及CRISPR/Cas9基因编辑株系的块根较野生型块根数显著增多，鲜重显著增加，淀粉含量显著提高。对IbCPK28及IbbZIP11转基因甘薯植株块根的RNA-seq及ChIP-seq数据进行联合分析，结果表明差异基因主要富集在植物激素信号途径、碳代谢、氮素转运等通路。同时，酵母双杂筛选获得一个E3泛素连接酶IbBRG2分别与IbCPK28和IbbZIP11相互作用。磷酸化实验表明IbCPK28能够磷酸化修饰IbBRG2，但不能磷酸化IbbZIP11。体外泛素化实验表明IbBRG2能够泛素化修饰IbbZIP11，但不能泛素化IbCPK28，且磷酸化的IbBRG2对IbbZIP11的泛素化修饰作用更强。推测IbCPK28被激活表达后，蛋白量增加，磷酸化IbBRG2程度增强，磷酸化的IbBRG2对IbbZIP11的泛素化降解增强，从而增强IbCPK28的转录，激活植物激素信号转导、碳水化合物合成与代谢、营养物质运输进而影响甘薯块根发育。研究为解决甘薯块根形成与发育的分子调控机制这一重要科学问题提供参考，同时为甘薯高产优质的分子育种提供新的基因。.此外，本项目资助申请人完成甘薯CDPK基因家族的全基因组鉴定及表达分析，IbBBX24-IbTOE3-IbPRX17作用模块通过调节活性氧途径调控甘薯逆境胁迫，IbBBX29调控甘薯类黄酮积累的分子机制等研究工作，研究结果发表于New Phytologist，Plant Physiology和International Journal Molecular Science等杂志，共发表论文7篇，其中SCI 论文 5 篇 ，会议论文2 篇；申请国家发明专利2项；培养了2名博士和2名硕士。圆满完成了研究目标。