木豆酚类活性成分胞外分泌相关ABC转运蛋白基因的筛选与功能研究

Cajanus cajan (L.) Millsp is a perennial woody plant that contains phenolics as the main bioactive ingredients with multiple medical and health benefits, which have become potential sources of new drugs and new chemical entities. The autotoxicity of phenolics makes them difficult to accumulate continuously in plant cells. ABC transporters can mediate the extracellular secretion of secondary metabolites, which is conducive to the continuous synthesis of metabolites and their subsequent recovery. In this project, the screening and function study of ABC transporter genes related to the extracellular secretion of phenolics in C. cajan was conducted. C. cajan cell culture system was established as the research platform. Through the transcriptome sequencing of excretion elicitor-induced cell samples, ABC transporter genes related to the extracellular secretion of phenolics were screened and cloned. Physical and chemical properties, subcellular localization and molecular evolution of the encoded proteins were analyzed. The C. cajan cell culture system that can overexpress key ABC transporter genes was built to achieve the extracellular secretion of phenolics and the continuous high yield. This project promotes the extracellular secretion of bioactive compounds from natural forest resources in the viewpoint of gene and molecule, which is in line with the emerging development trend of the modern forest chemistry combined with the molecular biology. More importantly, this project provides a technical system of C. cajan cell culture with high productivity of bioactive phenolics for pharmaceutical and chemical fields, which can get rid of the traditional way to plunder natural resources, and promote the development of green emerging industries in forestry fields.

木豆是多年生灌木植物，酚类化合物是其主要活性成分，具有多种保健和医疗功效，是众多新药和新化学实体的潜在来源。酚类化合物的自毒作用致使其在植物细胞中难以持续积累，ABC转运蛋白可介导次生代谢产物的胞外分泌，利于产物连续合成及后续回收。本项目将对木豆酚类活性成分胞外分泌相关ABC转运蛋白基因进行筛选和功能研究，建立木豆细胞培养体系为研究平台，通过分泌因子诱导样品的差异转录组测序，筛选并克隆与酚类化合物分泌相关的ABC转运蛋白基因，解析其编码蛋白理化结构特性、亚细胞定位和分子进化信息，构建过表达关键ABC转运蛋白基因的细胞培养体系，实现木豆酚类活性成分的胞外分泌和持续高产。本项目从基因分子角度促进林源活性成分的胞外分泌，符合现代林产化学与分子生物学相结合的新兴发展趋势，同时可为医药和化工领域提供胞外高产酚类活性成分的细胞培养技术体系，摆脱从自然资源中获取的传统方式，推动我国林业绿色新兴产业发展。

木豆是世界范围内重要的药食两用木本豆科植物，其富含的酚类化合物具有多种医疗和保健功效。植物离体细胞培养技术已成为生产具有药用活性/保健价值次生代谢产物的重要手段，但某些次生代谢产物因其自毒作用难以在细胞中持续积累。本项目以筛选木豆酚类活性成分胞外分泌相关ABC转运蛋白基因为导向，重点完成了以下研究内容：优化并建立了可精确定性和定量分析木豆中11个目标酚类活性成分的UPLC-MS/MS方法，为灵敏追踪木豆离体细胞培养体系中痕量级目标活性成分提供了高效的检测技术手段；建立了稳定可靠的木豆愈伤组织诱导和增殖培养体系，筛选到了高产愈伤组织细胞株系，优化并建立了生物量和目标活性成分产量高的木豆细胞液体悬浮培养体系，用以体外生产目标酚类活性成分以及作为研究其生物合成及胞外分泌的模式平台；重点开展了活体食用曲霉属真菌对木豆悬浮细胞的诱导排泌研究，优化并确定了目标酚类活性成分诱导增量及胞外分泌的最佳条件，发现米曲霉Y-29诱导可促使木豆细胞中两种重要酚类活性成分木豆芪酸和染料木素的胞外分泌量分别增加18.02倍和3.58倍；通过相关软件分析木豆中ABC转运蛋白的理化性质及保守区域进化关系，挖掘到与次生代谢产物转运相关的候选ABC转运蛋白基因24个，并初步明晰了介导木豆芪酸和染料木素胞外分泌的潜在关键ABC转运蛋白基因4个，并完成了其中1个基因(ABCB11)的序列全合成及过表达载体构建。研究结果为未来利用细胞悬浮生物反应器技术规模化生产木豆酚类活性成分奠定了基础，同时也为后续利用基因工程技术手段高效生产木豆重要酚类活性成分提供了理论参考。