棉籽油脂合成与调节关键基因的表达分析与功能验证

This research based on the pathway analysis of lipid synthesis of higher plants，several key genes involved in lipid synthesis and metabolism regulation were selected to identify the molecular regulation mechanism of lipid synthesis and metabolism in upland cotton. The genes included 1)encoding genes GhACCase, which determined carbon flux flowing into different metabolism pathways of either the lipid synthesis and protein synthesis; 2)encoding gene GhFAD2-1,which influenced the content of oleic acid, and 3)transcription factors GhDof1 and GhWRI which regulated lipid synthesis and responded to abiotic stress. Expression characterization of the target genes were checked in upland cotton during the process of fatty acid accumulation and lipid synthesis in different developmental stage of immature embryos. Over-expression vectors were constructed for GhAccase, GhDof1 and GhWRI1, and RNAi vectors were constructed for GhFAD2-1. Transgenic plants of upland cotton, Arabidopsis and tobacco were obtained, and the genetic model was analyzed using the transgenic plants of both Arabidopsis and tobacco. Furthermore, the transgenic plants of upland cotton with target genes of single copy should be identified, and self-pollinated to obtain the homozygotes, while intercrossed to obtain the materials to pyramided the genes. The expression profiles of target genes were identified in the individuals of transgenic plants and control to elucidate the function of genes combining the results of transformation of the all vectors. The mechanism to improve the quantity and the quality of lipid in upland cotton will be deduced. And the genetic modified stocks of upland cotton with the lipid altered in cottonseed will be obtained for further research.

本研究基于对高等植物油脂合成途径的分析，选择在脂肪酸合成起关键作用的编码基因GhACCase，参与调控油份合成的转录因GhDof1、GhWRI1，以及影响油酸含量的基因GhFAD2-1，研究其在棉花幼胚发育不同时期的油脂积累过程中的变化。基于分子克隆，构建GhACCase、GhDof1与GhWRI1过量表达载体、GhFAD2-1干涉载体，转基因获得了转基因棉花、拟南芥与烟草材料，分析转基因植物的遗传方式；鉴定出单拷贝的转基因棉花阳性植株，自交筛选获得纯合材料，杂交聚合多个基因；结合各载体转化分析结果和转录组测序结果，分析目的基因表达情况，研究油脂合成的分子调控机理，探明提高棉花油份含量、改善品质的机制；同时，分析转录因子GhDof1、GhWRI1的非生物胁迫表达模式，为棉花油份改良与抗逆育种奠定理论与技术基础。

棉花是重要的油料作物。棉籽含有丰富的脂肪酸，可用于加工食用油、工业原料和生物柴油。种子油脂合成代谢过程调控策略有：调节碳源分配，抑制碳源流入蛋白质合成路径的关键酶编码基因的表达、增强碳源流入脂肪酸合成路径的关键酶编码基因的表达；干预油脂合成，促进脂肪酸生物合成，提高三酰甘油组装过程的关键酶编码基因的表达水平；提高种子油脂的品质：改变植物脂肪酸组成，调节脂肪酸脱氢酶基因的表达；引入外源超长链多不饱和脂肪酸合成途径；调控油脂合成代谢途径的转录因子表达，提高种子油脂含量。.本研究开展工作包括调节碳源分配、调控油脂合成代谢途径的转录因子、棉籽表达谱分析等，以及油份代谢相关基因参与逆境胁迫的作用。棉籽发育不同时期转录组数据，分析揭示了棉籽油分合成调控的基因表达谱；研究了油分合成关键基因在棉籽发育、油分积累过程的表达模式，及其在逆境胁迫下的表达变化。棉花异质型GhACCase的四个亚基在棉籽发育过程的表达分析及其过表达分析，揭示了四亚基基因对棉籽油分含量的影响。通过对干涉GhPEPC转基因与对照材料的转录组比较分析，揭示了干涉GhPEPC基因提高棉籽油分的分子机理。克隆和分析了两个油分合成相关转录因子GhWRI1和GhDof1，并对其启动子区进行分析验证。此外，对棉籽油分含量和籽指进行QTL分析，发现在多环境下稳定的棉籽油分QTL 9个，可以应用于分子标记辅助选择育种。通过调节植物种子油脂代谢过程，是提高植物油脂含量和品质的有效途径。.项目彻底解决了3个拟“拟解决的关键科学问题”的第1和第3个问题，部分解决了第2个问题。基因聚合研究协同表达对油脂合成的影响问题还没有彻底解决：ACCase双亚基聚合没有显著提高棉籽油分含量，推测可能需要四亚基的协同表达。目前四亚基聚合刚得到F1代。.