Table_5_Genome-wide identification and comprehensive analysis of tubby-like protein gene family in multiple crops.xlsx

IntroductionThe highly conserved tubby-like proteins (TLPs) play key roles in animal neuronal development and plant growth. The abiotic stress tolerance function of TLPs has been widely explored in plants, however, little is known about comparative studies of TLPs within crops.
MethodsBioinformatic identification, phylogenetic analysis, Cis-element analysis, expression analysis, Cis-element analysis, expression analysis and so on were explored to analysis the TLP gene family of multiple crops.
ResultsIn this study, a comprehensive analysis of TLP genes were carried out in seven crops to explore whether similar function of TLPs in rice could be achieved in other crops. We identified 20, 9, 14, 11, 12, 35, 14 and 13 TLP genes in Glycine max, Hordeum vulgare, Sorghum bicolor, Arabidopsis thaliana, Oryza sativa Japonica, Triticum aestivum, Setaria italic and Zea mays, respectively. All of them were divided into two groups and ten orthogroups (Ors) based on amino acids. A majority of TLP genes had two domains, tubby-like domain and F-box domain, while members of Or5 only had tubby-like domain. In addition, Or5 had more exons and shorter DNA sequences, showing that characteristics of different Ors reflected the differentiated function and feature of TLP genes in evolutionary process, and Or5 was the most different from the other Ors. Besides, we recognized 25 cis-elements in the promoter of TLP genes and explored multiple new regulation pathway of TLPs including light and hormone response. The bioinformatic and transcriptomic analysis implied the stresses induced expression and possible functional redundancy of TLP genes. We detected the expression level of 6 OsTLP genes at 1 to 6 days after seed germination in rice, and the most obvious changes in these days were appeared in OsTLP10 and OsTLP12.
DiscussionCombined yeast two-hybrid system and pull down assay, we suggested that the TLP genes of Or1 may have similar function during seed germination in different species. In general, the results of comprehensive analysis of TLP gene family in multiple species provide valuable evolutionary and functional information of TLP gene family which are useful for further application and study of TLP genes.

引言 高度保守的类Tubby蛋白（tubby-like proteins, TLPs）在动物神经元发育与植物生长过程中发挥关键作用。目前学界已在植物中广泛研究了TLPs的非生物胁迫耐受功能，但针对作物内TLPs的比较研究仍相对匮乏。
方法 本研究通过生物信息学鉴定、系统发育分析、顺式作用元件分析、表达分析等多种手段，对多种作物的TLP基因家族开展分析。
结果 本研究针对7种作物的TLP基因开展了全面分析，旨在探究水稻中TLPs的功能是否可在其他作物中重现。本研究分别在大豆（Glycine max）、大麦（Hordeum vulgare）、高粱（Sorghum bicolor）、拟南芥（Arabidopsis thaliana）、粳稻（Oryza sativa Japonica）、普通小麦（Triticum aestivum）、谷子（Setaria italic）以及玉米（Zea mays）中鉴定到20、9、14、11、12、35、14和13个TLP基因。基于氨基酸序列，所有TLP基因可被划分为2大类群与10个正交群（orthogroups, Ors）。绝大多数TLP基因包含两个结构域：类Tubby结构域与F-box结构域，而Or5家族成员仅含有类Tubby结构域。此外，Or5所含外显子数量更多、DNA序列更短，表明不同正交群的特征反映了TLP基因在进化过程中的功能分化与特性差异，其中Or5与其他正交群的差异最为显著。本研究还在TLP基因的启动子区域鉴定到25个顺式作用元件，并揭示了TLPs受光与激素响应等多条全新调控通路。生物信息学与转录组分析结果显示，TLP基因存在胁迫诱导表达模式与潜在的功能冗余性。此外，本研究检测了水稻中6个OsTLP基因在种子萌发后1至6天的表达水平，发现OsTLP10与OsTLP12在该时段内的表达变化最为显著。
讨论 结合酵母双杂交系统与pull down实验结果，本研究推测Or1家族的TLP基因在不同物种的种子萌发过程中可能具有保守功能。总体而言，本研究对多物种TLP基因家族的全面分析结果，为TLP基因家族提供了极具价值的进化与功能信息，可为后续TLP基因的相关研究与应用提供重要参考。