棉花DPC敏感相关基因的定位、鉴定及响应DPC调控的分子机制研究

Mepiquat chloride is the widely used plant growth retardant to control excessive vegetative growth and coordinate the relationship between vegetative growth and reproductive growth in Xinjiang. In cotton breeding process, we found that the DPC sensitivity showed a great difference among different cotton varieties. It is not clear that which genes determine the DPC sensitivity of cotton and how they work. The study of this problem will not only help us to clarify the molecular mechansim of cotton response to DPC chemical control, but also provide a theoretical basis for the rational use of DPC in cotton production. In this study, RNA-seq approach will be used to analyze the gene expression changes in DPC sensitive and insensitive parents treated with DPC, and find the DPC sensitive genes and other genes related to cotton response to DPC. Simultaneously, we will construct DPC sensitive and insensitive pools, which are composed of DPC sensitive and insensitive individuals from a F2 population produced by a hybrid cross of DPC sensitive × insensitive varieties. Subsequently, the pools will be subjected to whole genome resequencing. Additionally, GWAS will be used to identify the DPC sensitive loci of cotton by using the data from whole genome resequencing of 338 natural populations. In this study, the RNA-seq, BSA whole genome resequencing and GWAS will be taken together to screen the DPC sensitive genes and other genes related to cotton response to DPC. Finally, we will clone the candidate genes and verified their functions, and elucidate the molecular mechanism of cotton response to DPC chemical control.

缩节胺（DPC）是一类抑制型植物生长调节剂，可有效控制作物的过度营养生长，协调营养生长和生殖生长间的关系，在新疆棉花生产中被普遍使用。在育种实践中，我们发现不同的棉花品种对DPC的敏感性存在很大的差异，棉花对DPC的敏感性是由哪些基因决定，又是如何起作用的尚不清楚。这一问题的阐明不仅有助于我们明确棉花响应DPC化控的分子机制，也为生产合理利用DPC提供理论基础。本研究拟以DPC敏感和迟钝品种为材料，进行DPC处理前后的转录组分析；利用DPC敏感和迟钝品种的F2分离群体构建DPC敏感池和迟钝池，对两个极端池及其亲本进行全基因组重测序；利用课题组前期338个品种的自然群体的全基因组重测序结果进行GWAS分析，鉴定棉花DPC敏感性关联位点。综合以上转录组测序、混池全基因组重测序和GWAS分析结果，筛选DPC敏感性相关基因和其它响应DPC的基因，克隆并研究其功能，明确棉花响应DPC化控的分子机制。

为了明确棉花DPC敏感和迟钝品种响应DPC后基因表达变化差异并定位DPC敏感性相关基因，本研究从棉花材料中筛选出DPC迟钝品种XLZ74和敏感品种SD1068。喷施DPC 1、3和6d后，分别收取两品种茎尖进行转录组测序；从两品种杂交F2群体中筛选DPC敏感和迟钝植株进行BSA混池测序，结合转录组测序结果定位DPC敏感性相关基因；利用VIGS和基因编辑技术对候选基因进行功能验证。. 转录组测序发现XLZ74与水对照间有6252个差异基因，SD1068有6163个差异基因。GO和KEGG分析表明两品种中激素合成和信号转导通路均受到影响。赤霉素降解基因GA2ox在两个品种中均明显上调表达，在敏感品种中上调幅度更为明显；对生长素信号起负调控作用的IAA/AUX和SAUR基因在两个品种中均上调表达，在敏感品种中较早上调表达；油菜素内酯生物合成相关基因仅在敏感品种中下调；大量转录因子在敏感品种中下调表达，而在迟钝品种中上调；另外，两品种经DPC处理后激素含量测定结果与转录组测序分析结果相符合。上述研究从分子水平解释了DPC对敏感品种抑制作用较强，而对迟钝品种抑制作用较弱的原因。. 根据BSA混池重测序结果，对SNP和InDel的关联区域取交集，在候选区域有1020个基因，包括非同义突变基因134个，移码突变基因6个。结合转录组测序结果，选取2个DPC敏感性相关基因GhTCP12和GhGA20ox1B进行功能验证。对两基因分别进行VIGS沉默研究，发现DPC处理后沉默植株株高均受到抑制，但差异不显著，表明抑制基因的表达导致棉花对DPC的敏感性下降。通过PCR检测，两基因共获得编辑植株9株，编辑类型主要为碱基缺失和错配。. 本研究为今后获得DPC敏感性种质材料及改良棉花栽培品种的DPC敏感性奠定了工作基础，同时为进一步阐明棉花响应DPC化控的分子机制及DPC在棉花生产中的有效应用提供理论基础。