茶树锌指蛋白CsDof1调控CsGs2介导谷氨酰胺合成的机理研究

Glutamine not only is a vital link to inorganic nitrogen assimilated to amino acid, but also plays a role in quality of tea (Camellia sinensis), but the regulation mechanism of glutamine biosynthesis is unclear. We already found that the transcription factor CsDof1 cloned from tea is not only a member of zinc finger protein, but also positively regulated CsGs2 at the transcription level. Moreover, CsGs2 promoted glutamine biosynthesis. Next, the overexpression of CsDof1 in the tea callus tissue will be determined to the biological function of CsDof1 both at the level of transcription and translation in the glutamine biosynthesis. Subsequently, we put forward clearly transcription and function domain of CsDof1 in vivo and in vitro, respectively. Finally, proteins interaction with CsDof1 will be screened , which will be confirmed by Co-immunoprecipitation, and function of them will be investgated, indicating that CsDof1 zinc finger protein regulated glutamine biosynthesis in tea. This study will lays a theoretical foundation for effective utilization of ammonium assimilation and improving quality of tea.

谷氨酰胺是无机氮同化为氨基酸的重要纽带，也是构成茶叶品质的重要游离氨基酸，但谷氨酰胺合成的调控机理不清楚。前期，我们发现CsDof1是一个锌指蛋白，并且在转录水平上正调控CsGs2的表达；此外，CsGs2促进谷氨酰胺合成。在此基础上，通过在茶树愈伤中过表达CsDof1，进一步从转录和翻译水平上鉴定CsDof1在谷氨酰胺生物合成途径中的功能。然后从体内和体外两个方面验证CsDof1与CsGs2启动子的结合，并明确结合的顺式作用元件，以揭示CsDof1调控CsGs2的分子机理。最后筛选与CsDof1互作的目的蛋白，并验证CsDof1与目的蛋白的互作关系，结合目的蛋白的功能分析，进一步明确CsDof1调控CsGs2介导谷氨酰胺合成的分子机理。为茶树氨同化的有效利用和茶叶品质遗传改良奠定理论基础。

谷氨酰胺是茶树氨同化的重要第一产物，也是构成茶叶品质的重要游离氨基酸，但谷氨酰胺合成的调控机理不清楚。本研究发现遮荫能够提高茶树叶片中谷氨酰胺的含量，CsDOF和CsGS2的表达量以及谷氨酰胺合成酶活性。茶树叶片CsDOF基因被抑制后，CsGS2的表达水平降低，谷氨酰胺含量减少。遮荫处理CsDOF沉默的茶树叶片，CsDOF基因的表达量和谷氨酰胺含量均高于日光照射的茶树叶片。同样，沉默茶树叶片CsGS2基因，CsGS2基因的表达量和谷氨酰胺含量均降低；遮荫处理CsGS2沉默的茶树叶片，CsGS2基因的表达量和谷氨酰胺含量均高于日光照射的茶树叶片。在拟南芥中异源表达CsDOF和CsGS2，CsGS2基因的表达量和谷氨酰胺含量均提高。酵母单杂交、LUC和EMSA试验证实CsDOF与CsGS2启动子结合，结合原件为‘AAAG’。茶树叶片中谷氨酰胺合成依赖于锌指蛋白CsDOF对谷氨酰胺合成酶CsGS2的调控作用。综上所述，本研究初步解析了茶树叶片谷氨酰胺合成的分子机理。为更好的研究茶树氨同化提供的理论基础。