棉花GaADC2在纤维起始中的功能研究

Cotton is an important cash crop in China, cotton fiber is an important raw material of textile industries. Our transcriptome analysis of fiber initiation material of Gossypium arboreum L. showed that the expression of arginine decarboxylase GaADC2 was significantly up-regulated, ADC is the key rate-limiting enzyme in the polyamine pathway, whereas there is no experimental evidence that ADC is involved in fiber development in cotton yet. In this project, the overexpression and interference lines of GaADC2 will be developed for illuminating its roles in fiber initiation, and analyzing the effects on its substrate arginine and downstream polyamine and hydrogen peroxide. The promoter of GaADC2 contains the essential cis-element of MYB transcription factor, DNA pull down, BLI, EMSA and ChIP techniques will be employed to clarify the binding of GaADC2 with interacting protein. The kinetics of arginine decarboxylation catalyzed by GaADC2 will be resolved by using prokaryotic expression system, and with addition of inhibitor D-arginine. The catalytic activity of GaADC2 and the rescue of putrescine defect phenotype will be identified in vivo through the transformation of Arabidopsis adc2-/- mutants. The function of GaADC2- mediated polyamines in fiber initiation will be analyzed by culturing cotton ovules in vitro, with addition of exogenous arginine, polyamine and inhibitors of polyamine, combined with RNA-Seq. These results will provide new evidence for the mechanism of GaADC2 regulation of fiber initiation, and lay theoretical foundation for the cotton fiber quality and quantity improvement through the genetic engineering way.

棉花是中国重要的经济作物，棉纤维是纺织工业最重要的天然纤维来源。本研究前期对亚洲棉纤维起始期材料转录组测序发现精氨酸脱羧酶GaADC2表达显著上升，ADC是多胺途径关键限速酶，目前尚未见棉花ADC参与纤维发育的实验证据。因此，本项目拟通过上调和抑制GaADC2的表达，分析其对底物和下游产物、过氧化氢含量的影响，研究其在棉花纤维起始中的功能。通过DNA pull down、BLI、EMSA和ChIP技术分析GaADC2启动子与MYB互作的调控机制。采用原核表达技术，并辅以D-arginine处理，研究GaADC2的反应动力学。转化拟南芥adc2-/-突变体，分析对缺陷表型的恢复。利用胚珠离体培养技术，外源添加多胺及抑制剂，结合RNA-Seq，分析GaADC2介导的多胺的功能。本研究将为解析GaADC2调控纤维起始的分子机理提供新的实验证据，也为基因工程技术改良棉花纤维产量和品质提供理论支撑。

棉花是重要的农作物，为纺织工业提供原料。纤维长度是直接影响纤维质量的关键因素。ADC (arginine decarboxylase，精氨酸脱羧酶)是多胺途径中的关键限速酶，但目前尚无实验证据表明ADC参与棉花纤维发育。我们对棉花纤维起始期的转录组分析表明，GaADC2的表达显著上调。在四倍体陆地棉(gosypium hirsutum L.)中，GaADC2的等位基因GhADC2在棉花纤维伸长过程中呈上调表达模式。多胺水平与纤维伸长相关，特别是ADC调节的腐胺量增加。扫描电镜显示，添加ADC底物或产物腐胺可增加纤维长度，而添加ADC特异性抑制剂可降低纤维密度。接下来，对外源添加腐胺的纤维材料进行转录组分析，共检测到差异表达基因3163个，主要参与苯丙类生物合成途径、脂肪酸延伸途径、倍半萜和三萜生物合成途径。转录因子MYB109、WRKY1和TCP14的编码基因富集。综上，本项目研究结果表明ADC2正向调控棉纤维伸长，为今后棉纤维发育的研究提供了基础。