菘蓝中靛蓝生物合成关键酶基因CYP的鉴定与功能研究

Indigo is the main secondary metabolites of Isatisindigotica Fort. The basic biosynthesis process of indigo is: indole→indoxyl→precursors of indigo→indigo. Among them, the CYP (cytochrome P450) is the key enzyme which oxidized indole to indoxyl by adding hydroxyl groups. This process is the branch point of synthesis of indigo in plant. By using Solexa deep sequencing technology, we obtained 51,698 unigenes from transcriptome of Isatis indigotica Fort..There are 23 enzymes participated in indigo biosynthesis, including 4 CYP genes which involved in hydroxylation of indole. In this proposal, we intend to analyze the relationship between the change of CYP gene expression and the accumulation of indigo in vivo under the biotic stress (fungal elicitors) and abiotic stress (light, drought, salt, etc.) conditions. By screening and cloning CYP genes, we also consider the characteristics of spatio-temporal expression of the CYP genes in vivo and confirm the functions of CYP gene in regulation of indigo synthesis process. In this study, we attempt to clarify the molecular mechanisms of indigo biosynthesis, and try to open up a new ways to produce indigo by bio-technology. Indigo is the main secondary metabolites of Isatisindigotica Fort. The basic synthesis process of indigo is: indole?indoxyl?precursors of indigo?indigo. Among them, the CYP (cytochrome P450) is the key enzyme which oxidized indole to indoxyl by adding hydroxyl groups. This process is the branch point of synthesis of indigo in plant. By using Solexa deep sequencing technology, we obtained 51,698 unigenes from transcriptome of Isatis indigotica Fort..There are23 enzymes participated in indigo biosynthesis, including 4 CYP genes which involved in hydroxylation of indole. In this study, we intend to analyze the relationship between the change of CYP gene expression and the accumulation of indigo in vivo under the biotic stress (fungal elicitors) and abiotic stress (light, drought, salt, etc.) conditions. By screening and cloning CYP genes, we also consider the characteristics of spatio-temporal expression of the CYP genes in vivo and confirm the functions of CYP gene in regulation of indigo biosynthesis process. In this proposal, we attempt to clarify the molecular mechanisms of indigo biosynthesis, and try to open up new thoughts and strategies to produce indigo by biotechnology.

靛蓝是菘蓝的主要代谢产物，其基本合成过程为：吲哚→吲哚酚→靛蓝前体→靛蓝。其中，吲哚在细胞色素P450单加氧酶的催化下加羟基形成吲哚酚，是吲哚形成靛蓝的重要分支过程。据此，我们推测编码细胞色素P450单加氧酶的CYP基因在靛蓝合成过程中具有重要作用。研究前期采用Solexa高通量测序技术进行菘蓝转录组de novo测序，共获得51,698 条Unigene，其中靛蓝合成相关基因23条，CYP基因4条。本研究拟通过分析生物（真菌诱导子）及非生物胁迫（光照、干旱、盐等）条件下CYP基因的表达变化与靛蓝积累之间的关系，筛选并克隆与靛蓝合成相关的CYP基因家族成员，进而研究其时空表达特性；采用过表达及基因敲除的方法，进一步阐明CYP基因在靛蓝生物合成过程中的作用。本项目预期研究结果将为深入探讨靛蓝生物合成的分子机制，利用生物技术生产靛蓝提供新的思路和策略。

通过本项目的研究，系统建立了菘蓝中靛蓝与靛玉红优化的超声提取条件，并在此基础上探讨了靛蓝与靛玉红在自然生长条件下、离体培养条件下，以及多种诱导子处理条件下的代谢积累规律，相关研究结果表明菘蓝各部位靛蓝与靛玉红的含量由高到低的顺序均表现为 叶 > 花 > 果 > 茎 > 根，除了根以外，菘蓝各部位靛玉红的含量高于靛蓝。离体培养条件下，菘蓝不定芽中靛蓝与靛玉红的含量高于愈伤组织，并接近于实生苗中二者的含量。不同种类诱导子（MJ、SA、YE、Cu2+、Ag+）处理均会对靛蓝与靛玉红的代谢积累产生一定的影响作用，并表现为不同的时间和浓度积累效应。结合高通量测序技术，依次探讨了对照与MJ、YE和Ag+三种诱导子处理条件下菘蓝叶的转录组差异，重点进行菘蓝CYP基因的鉴别与表达分析，相关研究结果表明，从菘蓝中共获得36,445个Unigene，筛选到291个CYP Unigene，并依次对其进行COG、GO、KEGG、KOG、Pfam、Swiss-Prot，以及NR的注释、分类与系统进化分析，以筛选与菘蓝次生代谢产物合成积累相关的CYP及其相关基因，并进一步克隆得到IiTSA、IiFeSOD、IiCHS和IiCYP83B1等基因。对菘蓝中7个CYP基因在不同诱导子处理条件下，以及菘蓝根、茎、叶中的表达水平进行了检测，以探讨菘蓝CYP基因的表达模式。最后，本项目将IiCYP83B1基因在菘蓝中进行干涉和过表达，并结合实时荧光定量PCR和高效液相色谱检测，比较干涉和过表达株系中靛蓝及相关代谢产物的含量水平，探讨IiCYP83B1基因在靛蓝及相关代谢产物合成积累中的作用，相关研究结果表明，IiCYP83B1基因在3个菘蓝转基因株系均有不同程度的表达，并以L3株系中的表达量最高，达到对照的近160倍；而靛蓝与靛玉红的含量较对照相比没有明显的升高，芥子油苷的含量则达到对照植株含量的2.27倍。总之，本项目较为系统研究了不同诱导子处理条件下菘蓝CYP基因的系统分类和表达情况，并进一步对菘蓝IiCYP83B1基因的功能进行了初步的探讨，相关研究结果对于了解菘蓝靛蓝、靛玉红与芥子油苷等次生代谢产物的合成积累过程提供了一定的参考和借鉴，同时也为进一步筛选和培育优良菘蓝种质资源提供了思路。