基于龙胆苦苷生物合成关键酶基因SNPs和CNVs的滇龙胆种质评价

The stability and controllability of Chinese herbal medicines can directly influence the effectiveness and safety of clinical drug use. Gentiana rigescens，as one of the original plants of Traditional Chinese Medicine“longdan”, mainly distributed in Yunnan. At present, the wild resources were lack and cultivated germplasm was confused. To reveal the genetic essence of quality difference, research on the functional genes is the most direct and key method. On the basis of high-throughput transcriptome sequencing and some other research, the project intends to clone three candidate key enzyme genes participating in gentiopicroside biosynthesis.The three enzyme genes include geraniol 10-hydroxylase, iridoid synthase and secologania synthas. Then, the key enzyme genes were determined by functional verification. Gene functions were detected by time-space of gene expression and in vitro enzymatic. On this foundation, The study focused on correlation between key enzyme gene SNPs and CNVs and gentiopicroside synthesis of three years of Gentiana rigescens, which collecting from different germplasm were planted in the same habitat. The study aimed at uncovering the genetic essence of quality difference in Gentiana rigescen. The results can provide the basis for evaluating the quality of Gentiana rigescens, but also lay the foundation for quality control.

中药材质量的稳定性和可控性直接影响临床用药的有效和安全。滇龙胆作为中药材龙胆的基源植物之一，主产云南，目前野生资源匮乏，栽培种质混杂不清，导致药材品质不稳定。若要揭示品质差异遗传本质，对功能基因的研究是最直接和关键的手段。本项目拟在高通量转录组测序和其他研究的工作基础上的，克隆参与滇龙胆的主成分龙胆苦苷生物合成的三个候选关键酶基因(香叶醇-10 羟化酶、环烯醚萜合酶、裂环烯醚萜合酶基因)，进一步通过对候选关键酶基因采用时空表达特性以及体外酶促反应进行功能验证确定关键酶基因。在此基础上，以同生境下栽种的不同种质滇龙胆为材料，研究三年生滇龙胆关键酶基因的SNPs和CNVs与龙胆苦苷积累的关系，以揭示滇龙胆品质差异遗传本质。研究结果可为滇龙胆的品质评价提供依据，也可为品质调控奠定基础。

作为中药材龙胆的基源植物之一滇龙胆，目前野生资源匮乏，栽培种质混杂不清，导致药材品质不稳定,揭示滇龙胆品质差异遗传本质，对于调控药材品质有重要意义。本项目克隆了三个关键酶基因(3-羟基-3-甲基戊二酰辅酶A还原酶HMGR、香叶醇-10羟化酶G10H、环烯醚萜合酶IS)，通过原核表达初步验证了三个基因的功能。测定了3个基因的根、茎、叶中于四个发育时期的表达量；并测定对应滇龙胆的不同部位于不同生长时期的龙胆苦苷含量。GRHMGR和GRG10H在滇龙胆四个发育时期的根、茎和叶中均有表达，GRIS只在滇龙胆四个发育期的茎和叶中检测到表达，推测龙胆苦苷可能由叶中合成经过茎运输到根中贮存。对G10H和SLS进行了体外酶促反应研究，初步验证了G10H和SLS基因的功能，为进一步研究G10H和SLS基因在环烯醚萜类生物合成途径中的具体机制奠定基础。研究了滇龙胆关键酶基因的SNPs和CNVs与龙胆苦苷积累的关系，发现滇龙胆样品G10H其编码区氨基酸变异位点较为丰富，按氨基酸序列279~339片段的明显差异可分为7类，产地之间变异明显; IS基因编码区氨基酸序列中共有16个变异位点，分型统计发现20种类型；三个基因均存在1、2和3拷贝，其中G10H基因拷贝数类型较为丰富，并出现4个及4个以上的多拷贝，IS基因主要为1拷贝，SLS基因主要为1拷贝，滇龙胆G10H基因1拷贝可能更有利于根中马钱酸和獐牙菜苦苷的积累，而多拷贝可能更有利于叶中龙胆苦苷的积累。本研究培养3名硕士研究生，发表论文5篇，其中4篇为中文核心期刊，一篇SCI审稿中。研究结果提供了滇龙胆生物合成途径的遗传基础，可为品质调控奠定基础。