陆地棉纤维发育相关TCP转录因子的挖掘和功能研究

Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play versatile functions in multiple aspects of plant growth and development. However, no systematical study has been performed to uncover the mechanism of this transcription family involved in fiber development. In this study, we performed for the first time the genome-wide identification of the TCP transcription factor family in Gossypium hirsutum. A total of 73 non-redundant cotton TCP encoding genes were identified by homologous comparison method. To get a better understanding of the evolutionary history and phylogenetic relationships, an unrooted phylogenetic tree was constructed on the basis of multiple sequence alignment between upland cotton TCP protein sequences and 24 Arabidopsis TCP protein sequences. Several fiber-specific TCP transcription factors were isolated based on the expression profiles analysis preliminarily. Chromatin Immunoprecipitation and Yeast two-hybrid analysis will be used to separate downstream target genes and interacting proteins in fiber. For further analysis of the gene function and regulatory mechanism, overexpression strategies and CRISPR/Cas genome editing system will be applied in cotton. Ultimatly, we hope to clarify the biological function and regulatory mechanisms of Gossypium hirsutum TCP transcription factors in fiber development, as well as excavate the key genes contributing to cotton fiber yield and quality improvement.

TEOSINTE-BRANCHED1/CYCLOIDEA/PCF（TCP）转录因子是一类植物所特有的转录家族，在植物生长发育的众多方面都发挥至关重要的作用。然而，关于TCP转录家族在纤维发育过程中的调控作用还没有系统的研究。本项目首次从基因组水平鉴定陆地棉TCP转录因子家族，利用同源比对法从陆地棉中分离到73个TCP转录家族成员。为了进一步了解基因家族的进化关系，分析陆地棉与拟南芥24个TCP蛋白之间的系统发育关系。根据组织表达模式分析初步筛选纤维不同发育阶段特异表达的TCP转录因子基因。通过染色质免疫共沉淀和酵母双杂交技术分离TCP转录因子的下游靶基因和互作蛋白。采取超量表达策略和CRISPR/Cas基因组编辑技术在棉花中进行转基因功能验证。最终，我们希望能够初步阐明陆地棉TCP转录因子在纤维发育过程中的生物学功能和分子作用机理，筛选关键功能基因用于棉花产量和品质分子改良。

棉花是世界上最重要的经济作物之一，其产物棉花纤维为纺织工业提供最主要的天然原材料。为此，阐明棉花纤维发育的分子机制已成为当前棉花研究中的主要目标。TCP转录因子是一类植物所特有的转录家族，通过转录激活或抑制在植物生长发育的众多方面都发挥至关重要的作用。然而，TCP转录家族的作用机理尤其在纤维发育过程中的调控作用还没有清楚的研究。本项目主要研究TCP转录因子对棉花纤维发育的影响和分子作用机制。从棉花中克隆到一个class I TCP转录因子GrTCP11，该基因与拟南芥AtTCP11高度同源，并在花药、纤维发育起始和次生壁合成阶段优势表达；在拟南芥中异源表达GrTCP11导致根毛长度变短；实时定量PCR分析发现转基因株系中茉莉酸合成和响应相关基因（AtLOX4, AtAOS, AtAOC1, AtAOC3, AtJAZ1, AtJAZ2, AtMYC2 and AtERF1）下调表达；HPLC-MS/MS测出转基因株系中JA含量相比野生型明显降低；表达量最高的转基因系4-1与突变体ataos3一样对50 μM MeJA不敏感。以上研究结果证明GrTCP11可能通过负调控茉莉酸合成和响应对影响纤维和根毛的发育。