大豆GmTCP670基因功能验证及表观修饰与其表达调控关系的研究

The TCP proteins are plant-specific transcription factors controlling diverse development and physiological processes. However, there is limited number of reports about the TCP transcription factors (TFs) in soybean. Our previous studies related to the molecular mechanism involved in the allergenic α-subunit-null improving hypoallergenic soybean seed protein quality have highlighted some unique features of soybean TCP TF GmTCP670 gene. For instance, using Map-Based-Cloning combined transcriptome RNS-Seq comparison approach, we recently identified a candidate α-null-related gene, its NCBI number is Gm20g28670 and was annotated as TCP TF, we designated it as ‘GmTCP670’. We have also identified that the methylation level of α-subunit gene promoter is correlation to the differential expression of GmTCP670. All of these data suggest that the differential expression of GmTCP670 is closely associated with the α-subunit deficiency. Therefore, understanding the gene function of GmTCP670 and its molecular mechanism of expression and regulation will be very useful for further explore the mechanism of allergenic-subunit-dificiency modifying the protein concentration and composition of hypoallergenic soybean. In this project plans, we are going to carry out the following researches: Yeast-One-Hybrid system will be used to identify whether GGNCCC TCP cis-motif related with GmTCP670. The possible protein interacting with GmTCP670 will been screened by Yeast-Two-Hybrid. Methylome and Transcriptome will be compared using the overexpression and CRISPR/Cas9 transgenic plants of T3 generation. The function of GmTCP670 will be analyzed comprehensively. All above work will help us to highlight the role of GmTCP670 in hypoallergenic soybean seed protein quality fromation.

本团队前期图位克隆与生物信息多组学联合分析的结果表明：TCP转录因子基因GmTCP670是大豆7Sα-亚基缺失相关基因，α-亚基基因启动子区的甲基化状态与GmTCP670基因的差异表达有关，该基因极可能参与了致敏蛋白α-亚基缺失引起的低致敏大豆蛋白成分重组及品质改良，本研究将首先通过转基因超表达和CRISPR/Cas9基因沉默技术，研究其基因功能；继而利用酵母单杂交、EMSA及ChIP分析法，验证GmTCP670的调控元件、确定其调控的靶基因；利用酵母双杂交技术鉴定与GmTCP670转录因子互作的蛋白，利用WGBS技术鉴定GmTCP670对大豆表观特性的影响，利用RNA-Seq技术解析GmTCP670基因的信号传导途径，系统阐明大豆TCP转录因子GmTCP670基因在‘α-缺失型低致敏大豆’亚基缺失及蛋白营养品质改良中的作用机制，初步了解表观修饰与GmTCP670基因表达调控的关系。

本项目前期研究结果证明：GmTCP670转录因子与大豆7S球蛋白α-亚基缺失及蛋白品质改良相关。本研究首先通过对GmTCP670基因的超表达(GmTCP670-OE)及CRISP/Case9 (GmTCP670-CC)基因编辑植株主要农艺性状（包括叶型、开花时间、株高，生育期）的差异比较，解析其基因功能；利用RNA-Seq及WGBS比较分析结果，鉴定GmTCP670基因超表达及基因沉默后转录组水平及全基因组甲基化水平的变化，解析GmTCP670基因主要调控的信号传导途径及其对大豆表观特性的影响。.转基因功能验证结果表明：与对照东农50（DN50）及CRISP/Case9编辑株系相比，GmTCP670基因超表达转基因株系开花时间延迟；叶片变大、叶型由受体亲本东农50的尖叶变为圆叶型。植株比同期亲本及GmTCP670-CC株系高大，成熟期晚13天。基因编辑后获得的GmTCP670-CC转基因株系的氨基酸总量、必须氨基酸总量及精氨酸含量均高于对照DN50及GmTCP670-OE株系。以上结果表明，GmTCP670转录因子具有调节大豆叶型、花期、生育期及氨基酸品质的功能。 .转录组KEGG差异比较分析结果表明：在GmTCP670-CC vs. DN50组中有7个显著富集的通路，其中富集分数最高的为“维他命B6代谢”通路；在GmTCP670-OE vs . DN50组中，共有35个显著富集的通路，富集分数最高的通路为“亚油酸代谢”通路。.综合全基因组甲基化差异比较分析结果，与对照DN50比较，我们发现转录组差异表达基因、同时发生甲级化修饰改变的基因共有13个，其中GmTCP670-CC vs. DN50组与氨基酸转运相关的基因共有2个；GmTCP670-OE vs . DN50组，与生长发育相关的基因共有4个；与氨基酸代谢相关的基因有4个；与精氨酸合成、代谢相关的基因有3个。.利用酵母单、双杂，验证GmTCP670的调控元件及互作蛋白，结果表明GmTCP670转录因子能够识别并结合GTGGNCCC元件；能够与Glyma18G297100编码的蛋白互作并激活报告基因的表达。本研究的结果为今后进一步深入研究GmTCP670基因对大豆发育及氨基酸品质改良的调控作用及其分子机制提供了转录组及表观遗传学的基因资源。