油梨果肉转录因子WRI1-like对酰基载体蛋白基因ACP4的转录调控机制研究

Avocado is the famous subtropical/tropical fruit crop in the world and enjoys the high reputation of “forest cream” because of a large number of fatty acids in mature pulp. At present, the biochemical pathway of fatty acid synthesis has been clearer, but the transcriptional regulatory mechanism is not yet understood. Transcription factor WRINKLED1-like regulated some genes encoding key enzymes of fatty acid synthetic pathway. Previously, the full-length of WRI1-like was cloned with RACE technique, and further qRT-PCR validated that WRI1-like and ACP4 were expressed in avocado pulp. The present project will further 1）the green fluorescent fusion protein technology and yeast two hybrid system will be used for charactering the biological functions of WRI1-like; 2）the regulation of ACP4 by WRI1-like will be confirmed by means of dual-luciferase, yeast one hybrid system, and transgenic technology; 3）in order to uncover the relevance between the dynamic expression of WRI1-like and ACP4 and the changes of the fatty acid compositions and oil droplets, qRT-PCR, gas chromatography-mass spectrometer, and laser confocal microscopy will be adopted. The research results will provide the new target gene for the fatty acid synthesis WRI1-like regulate and enrich the transcriptional regulatory mechanism of fatty acid synthesis in avocado pulp.

油梨是世界著名的热带、亚热带果树，其成熟果肉中含有大量脂肪酸，因而享有“森林奶油”的美誉。目前，脂肪酸合成的生化途径已比较清楚，但其转录调控机理尚不明确。转录因子WRINKLED1-like调控一些脂肪酸合成途径关键酶基因。项目前期采用RACE技术克隆了WRI1-like全长，并通过qRT-PCR进一步验证了WRI1-like和ACP4在油梨果肉中的表达。本项目将进一步1）运用绿色荧光融合蛋白技术和酵母双杂交体系，分析WRI1-like生物学功能；2）采用双荧光素酶体系、酵母单杂交体系和转基因技术，明确WRI1-like对ACP4的调控；3）基于qRT-PCR、气质联用和激光共聚焦显微技术，明确油梨果肉发育过程中WRI1-like、ACP4动态表达与脂肪酸成分含量和油体变化的关联性。研究结果将为油梨果肉WRI1-like调控脂肪酸合成提供新的靶基因，丰富油梨果肉脂肪酸生成的转录调控机制。

油梨是世界著名的热带、亚热带果树，其成熟果实中含有大量脂肪酸，但其脂肪酸合成转录调控机理尚不明确。本项目1）通过转录组测序、qRT-PCR、脂肪酸含量检测和油体细胞学观察，阐明了果肉与种子发育期脂肪酸含量差异的机制，同时，明确了油梨果肉发育过程中PaWRI1-2动态表达量与PaACP4-2动态表达量、脂肪酸含量和油体体积呈正相关；2）为更好的理解PaWRI1-2的生物学功能，我们对PaWRI1-2所属的AP2/ERF转录因子家族进行进化分析，明确了可能与脂肪酸合成相关的成员信息；3）分别创制含有油梨果肉PaWRI1-2与PaWRI2的转基因番茄，通过转录组测序、qRT-PCR和脂肪酸含量检测，间接证明只有油梨果肉PaWRI1-2促进脂肪酸含量积累；同时，通过转录组测序，发现油梨果肉PaWRI1-2促进SlACP4表达上调；4）采用绿色荧光融合蛋白技术，确定PaWRI1-2蛋白定位于细胞核；采用酵母双杂交体系，明确PaWRI1-2具有转录自激活活性；采用双荧光素酶和酵母单杂交体系，确定PaWRI1-2蛋白与PaACP4-2启动子存在互作关系。该研究明确了PaWRI1-2通过介导脂肪酸合成通路中的功能基因（包括PaACP4-2）促进油梨果肉脂肪酸积累，增进了我们对油梨果肉脂肪酸生成的转录调控机制的理解。