MATE蛋白介导辣椒素液泡跨膜转运和积累的分子机制解析

辣椒素是辣椒中特有的一种生物碱类辣味物质，是其果实品质标志性状。辣椒素只在果实胎座细胞的细胞质中合成，却在液泡中大量积累；辣椒素由细胞质向液泡的跨膜转运是其转运、积累的第一步，是消除产物反馈抑制，促进辣椒素生物合成和积累的关键环节。MATE蛋白是多种生物碱跨膜转运的转运子，但其在辣椒素转运中的功能还未见报道，辣椒素跨膜转运过程及相关分子机理的研究尚属空白。课题组前期研究发现2个MATE蛋白能在体外跨膜转运辣椒素，其表达抑制能显著降低胎座辣椒素的含量，说明MATE可能是辣椒素跨膜的关键转运子。本项目将通过辣椒素分子荧光示踪技术进一步厘清辣椒素在胎座细胞内的转移轨迹；采用爪蟾卵母细胞系统分析MATE转运辣椒素的特性；利用CRISPR/Cas9等技术鉴定MATE基因功能。解析MATE蛋白介导辣椒素跨膜转运的分子机制及其与辣椒素生物合成之间的互作关系，为辣椒素积累调控及辣椒品质改良提供理论依据。

Capsaicin is an alkaloid spice only produced in capsicum plants, and is also an important quality character of Capsicum fruit. Capsaicin is only synthesized in the cytoplasm of fruit placenta cells, but accumulated in vacuoles. The transmembrane transport of capsaicin from cytoplasm to vacuoles is the first step of capsaicin transport and accumulation, and is the key link to promote the process of biosynthesis and accumulation of capsaicin, while eliminating inhibition of product feedback. MATE proteins are the transporter of many alkaloids transported across the membrane, but its function in capsaicin transport was never reported. The process of capsaicin transmembrane transport and related molecular mechanism remain unknown. In the present project, two MATEs were found to be able to transport capsaicin across the membrane in vitro, and the inhibition of their expression can significantly reduce the capsaicin content of placenta, indicating that these MATEs may be the key capsaicin transporter across membrane in Capsicum plants. This project will further clarify the transfer traces of capsaicin in placenta cells by capsaicin fluorescence tracing technology, analyze the characteristics of MATEs in capsaicin transport by Xenopus oocyte system, and identify the function of MATE genes by CRISPR/cas9 and other technologies. The aim of this project is to elucidate the molecular mechanism of MATE proteins in capsaicin transmembrane transport and the interaction between vacuole transport and biosynthesis of capsaicin. The results from this project will provide theoretical basis for the generation of pepper cultivars with expected fruit pungency levels by genetic engineering and breeding.