GhMYC2-like调控棉花色素腺体发育的分子机理研究

Utilization of cottonseed oil and protein are seriously restricted by toxic gossypol. There is no gossypol in glandless cotton. Therefore, it will be of great importance in genetic engineering breeding of low-gossypol cotton to reveal the gene regulatory network of pigment gland development. In our previous study, through map-based cloning, we have obtained a transcription factor GhMYC2-like and confirmed it as a vital positive regulator of pigment gland development. By transcriptome analysis, 504 genes co-expressed with GhMYC2-like were identified as its candidate target genes. This project will conduct chromatin immunoprecipitation sequencing (ChIP-Seq) assay to analyze the DNA binding sites of GhMYC2-like transcription factor and identify the genes whose promoter interact with GhMYC2-like. Target genes of GhMYC2-like will be identified by combining the results of ChIP-seq with transcriptome analysis. Then, ChIP-qPCR, electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay will be carried out to determine the target genes of GhMYC2-like. And, the function of GhMYC2-like target genes will be characterized in pigment gland development through gene knockout and overexpression analysis. These results would discover the molecular mechanism of GhMYC2-like regulating the pigment gland development. This project will provide the theoretical basis for illuminating the regulatory mechanisms of the pigment gland development in cotton and for low-gossypol cotton breeding.

有毒的棉酚限制了棉籽油和蛋白的利用，而无色素腺体的棉花不含有棉酚，研究色素腺体发育相关基因及其调控网络对低酚棉的基因工程育种具有重要意义。前期研究中，我们通过图位克隆得到了GhMYC2-like转录因子，证实它是色素腺体发育的关键正调控因子。通过转录组分析，将504个与GhMYC2-like共表达的基因锁定为其候选靶基因。本项目拟通过染色质免疫共沉淀测序（ChIP-seq）分析GhMYC2-like的DNA结合位点，筛选启动子区与GhMYC2-like相互作用的基因，结合转录组分析结果，鉴定GhMYC2-like的靶基因。利用ChIP-qPCR、凝胶阻滞电泳分析（EMSA）和双萤光素酶实验对靶基因进行确认，并通过基因敲除和过表达分析靶基因在色素腺体发育中的作用，揭示GhMYC2-like调控棉花色素腺体发育的分子机理。本研究将为解析色素腺体发育的分子机制和低酚棉育种提供重要的理论依据。

色素腺体中有毒的棉酚限制了棉籽产品的利用，对色素腺体发育的研究对棉花的综合利用具有重要意义。GhMYC2-like是色素腺体发育的关键调控因子，但是其调控机理还不清楚。本项目利用染色质免疫共沉淀测序（ChIP-seq）和转录组测序（RNA-seq）筛选GhMYC2-like的下游基因，并通过病毒诱导的基因沉默（VIGS）对下游基因进行了功能分析。共筛选到20个在有腺体和无腺体材料之间差异表达的候选基因，功能分析发现其中5个基因沉默后导致腺体数量不同程度减少。亚细胞定位分析表明，5个基因编码蛋白中2个核定位、2个膜定位和1个核膜共定位，暗示它们可能在色素腺体发育中发挥不同的作用。同时，我们鉴定到一个GhERF105基因受GhMYC2-like调控，该基因在无腺体材料各个组织中表达量均显著低于有腺体材料，沉默该基因可导致色素腺体数量和棉酚含量均降低。亚细胞定位表明GhERF105位于细胞核，并且可能作为转录激活因子通过结合GCC-box和DRE顺式作用元件调控下游基因。此外，我们还筛选到GhMYC2-like下游的6个NAC基因与色素腺体发育相关。同时，另一个MYC转录因子CGF1可能通过影响GhMYC2-like和6个NAC基因的表达来参与色素腺体发育的调控，从而构成一个MYC-NAC调控网络。本项目研究为深入解析GhMYC2-like的调控机理和阐明色素腺体发育机制奠定了基础，同时为低酚棉育种提供了理论参考。