油桐FATB基因油脂合成期促进α-桐酸高效积累的分子机制研究

Alpha-eleostearic acid is the main component in tung oil and also is an important quality index of tung oil as drying oil. The cultivation of tung tree varieties with high content of α-eleostearic acid is the main objective of tung tree breeding. The development of tung oil industry is seriously hindered because the genic regulation mechanism of α-eleostearic acid synthesis specifically catalyzed by tung tree is still unknown. Our preliminary study showed that FATB gene of tung tree promoted α-eleostearic acid accumulation through up-regulating expression during oil synthesis stage in kernel and FATB gene very likely located in the upstream of FADX gene to supply free dienoic acid for α-eleostearic acid synthesis. In view of this, this research will firstly elucidate the location and function of tung tree FATB in α-eleostearic acid synthesis pathway through methods such as analysis of free fatty acid profiles in Escherichia coli expressing tung tree FATB gene and analysis of substrate specificity of FATB. Given that significantly up-regulated expression of FATB gene during oil synthesis stage, this research will also reveal the expressive regulation mechanism of FATB gene through methods such as transcription factor screening, binding activity analysis between transcription factor and promoter sequence and transcription complex composition analysis. Finally, through comprehensive analysis of the results obtained from mechanic researches on gene function and expressive regulation, this study will clarify the molecular mechanism of α-eleostearic acid synthesis regulated by FATB gene in order to promote the understanding of synthesis mechanism of high content of α-eleostearic acid in tung oil.

α-桐酸是桐油主要成分，也是桐油作为干性油的重要质量指标。培育高α-桐酸含量油桐品种是油桐育种的主攻目标，但目前尚不清楚油桐特异性催化α-桐酸形成的基因调控机制，这严重阻碍了桐油产业的发展。前期研究发现，油桐FATB基因在种仁油脂合成期上调表达促进α-桐酸形成，且其很可能定位于FADX基因上游，为FADX提供合成α-桐酸的游离二烯酸。因此，本项目首先将通过转油桐FATB基因大肠杆菌的游离脂肪酸谱分析、FATB底物特异性分析等研究方法，明确油桐FATB在α-桐酸形成通路中的位置与功能。鉴于FATB基因在油脂合成期显著上调表达，本项目还将通过基因转录因子筛选、转录因子的启动子结合活性分析、转录复合体构成分析等研究方法，揭示FATB基因的表达调控机理。最后，通过综合分析基因功能和表达调控机理研究结果，本项目将阐明FATB基因调控α-桐酸形成的分子机制，以促进对桐油中高α-桐酸含量形成机理的认识。

桐油中α-桐酸含量高达70%以上，这表明油桐中某些脂肪酸合成途径基因可催化α-桐酸的形成，研究这些基因的功能是揭示油桐α-桐酸形成机理的基础。项目组前期研究发现，油桐FATB基因在油脂合成期上调表达，且其表达量与α-桐酸的含量正相关，进一步对转油桐FATB基因的拟南芥植株中脂肪酸种类和含量进行分析发现，FATB基因很可能定位于FADX基因上游，为FADX酶提供合成α-桐酸的游离二烯酸。为了验证这一科学推断，本项目通过体外催化体系的构建、催化活性分析和游离脂肪酸谱分析等方法，明确了FATB酶在油桐α-桐酸形成通路中的定位，为阐明油桐FATB基因促进α-桐酸形成的分子机理提供理论参考，并为油桐α-桐酸形成通路的完善提供了数据支撑。基因表达模式和蛋白定位研究发现，FATB基因的表达量与油桐种子中的油脂含量正相关，FATB蛋白定位于叶绿体。表型互补研究发现，油桐FATB基因能够恢复拟南芥fatb突变体的表型。将油桐FATB基因在大肠杆菌和拟南芥fatb基因突变体中过表达，导致大肠杆菌和拟南芥突变体中18:0和18:3脂肪酸含量的增加。分别用油桐种子的提取液和纯化的油桐FATB蛋白进行硫酯酶活性分析发现，只有18:0-ACP是油桐FATB的底物。以上结果表明，油桐FATB通过水解18:0-ACP，为下游相关酶提供底物，促进18:3脂肪酸的合成，以满足正常的生长发育所需。