转录因子GhNST3-A12调控棉花纤维发育机理的解析

In this study, we found a transcription factor based on our previous RNA-seq data, showing specific expression in developmental fiber. Realtime PCR results showed that relative expression level of GhNST3-A12 peaked at 20 DPA developmental fibers, which is a critical period of fiber rapid elongation and secondary cell wall synthesis. Further physical mapping showed that GhNST3-A12 was co-localized with QTL regions of fiber related traits, mainly micronaire QTLs and fiber length QTLs. It was speculated that GhNST3-A12 was the candidate gene. Moreover, GhNST3-A12 overexpressing Arabidopsis plants exhibited small rosette size and stunted growth of leaves with upward-curling blade, which was similar with that of AtNST3. So, we cloned GhNST3-A12 gene in cotton, carrying out research work by methods of overexpressing and CRISPR. The goal of this research is to clarify the relationship between GhNST3-A12 and developmental process of fiber. By screening GhNST3-A12 downstream target genes, we will further clarify the mechanism of GhNST3-A12 in controlling fiber development.

本研究利用实验室前期的RNA-seq表达数据，筛选到一个在棉花纤维中特异表达的转录因子，Realtime PCR结果表明，GhNST3-A12在20 DPA的纤维中表达量最高，该时期为纤维快速伸长和次生壁合成的关键时期。经过定位分析发现，GhNST3-A12定位于A12号染色体棉纤维性状QTL区间内，主要为纤维马克隆值QTLs和纤维长度QTLs，推测GhNST3-A12可能为其候选基因。转基因拟南芥结果表明，超表达株系表现出莲座叶变小，叶片向上卷曲的表型，与拟南芥次生壁调控基因AtNST3表型类似。本研究克隆棉花GhNST3-A12基因，采用过表达转基因和基因组编辑CRISPR手段，获得转基因GhNST3-A12和突变体ghnst3-a12的棉花材料，鉴定GhNST3-A12调控纤维性状的功能。并进一步筛选GhNST3-A12的下游靶基因，阐明GhNST3-A12调控棉纤维发育的分子机理。

棉花是一种重要的经济作物，是最重要的天然纤维来源。近年来，我国棉花生产表现为棉花种植面积不断下滑，棉花纤维品质较差等特点，改良纤维品质成为棉花育种的重要目标，虽然调控棉花纤维发育的功能基因越来越多，但是距离阐述清楚棉花纤维发育的生物学过程还远远不够。本研究利用实验室前期的RNA-seq表达数据，筛选到一个在棉花纤维中特异表达的转录因子GhNST3-A12，Realtime PCR结果表明，该基因在20 DPA的纤维中表达量最高，该时期为纤维快速伸长和次生壁合成的关键时期。转基因拟南芥结果表明，过表达株系表现出莲座叶变小，叶片向上卷曲的表型，与拟南芥次生壁调控基因AtNST3表型类似。转基因棉花结果表明，过表达株系表现出纤维变短、次生壁加厚的表型。构建GhNST3-A12和GhNST3-D12基因的CRISPR敲除载体，筛选到棉花CRISPR敲除纯合株系多个，CRISPR敲除株系表现出纤维变长、次生壁厚度降低的表型。利用野生型、超表达转基因材料和突变体材料进行差异表达基因分析，分析到一系列差异表达的候选基因，结果表明GhNST3-A12正调控次生壁合成相关基因的表达。本研究对棉纤维品质基因工程改良提供重要理论依据和基因资源。