基于CRISPR/Cas9技术解析白木香转录抑制因子AsJAZ对沉香倍半萜合成的调控机制

Generally, healthy trees of Aquilaria sinensis will not produce agarwood, but wounding of the tree initiates the sesquiterpenes biosynthesis and production of agarwood. Our findings demonstrate for the first time that jasmonate signal pathway is an important postwounding signal in A. sinensis that leads to the induction of sesquiterpene biosynthesis. Overexpression of AsJAZ of jasmonate signal pathway from A. sinensis markedly inhibited the expression of the sesquiterpene synthase genes in Arabidopsis thaliant. To verify the hypothesis that AsJAZ is likely to block the sesquiterpene gene expression as a negative regulator, this study will be carried out as follows. Firstly, isolation and function analysis genes of AsJAZ based on the data of genome of A. sinensis will be done. And the genome-wide expression profiles of AsJAZ genes will be investigated using the Solexa/Illumina digital gene expression (DGE) system, a tag based high-throughput transcriptome sequencing method, and the developmental processes which occur during early agarwood formation were systematically analyzed. Secondly, The CRISPR/Cas9 plant expression vector will be constructed to obtain the jaz knockout mutations of A. sinensis. Thirdly, the regulation mechanisms of AsJAZ in sesquiterpenes biosynthesis were vitrified using qRT-PCR and GC-MS under wounding stress and signal treatment, respectively. Finally, screening the jaz mutant with high sesquiterpene components and regulation mechanisms of the transcriptional repressor AsJAZ Proteins in sesquiterpenes biosynthesis will be confirmed. This project will provide basic data for rapid knockout of gene clusters that control the adverse traits of medicinal plants and to achieve rapid genetic improvement. And it also will provide a theory basis for medicinal plants with high medicinal components in novel germplasm innovation and crop breeding improvement.

珍稀濒危白木香只有受到伤害才产生沉香倍半萜并积累形成沉香。前期项目组证实伤害通过茉莉酸信号途径调控沉香倍半萜合成，在拟南芥中过表达白木香茉莉酸信号途径的AsJAZ蛋白显著抑制拟南芥倍半萜合酶基因表达。为验证“AsJAZ作为转录抑制因子阻遏沉香倍半萜基因表达”假说，本项目拟首先从已建立的白木香全基因组序列图谱中分离鉴定AsJAZ基因家族成员并通过数字基因表达谱技术揭示其伤害应答机制；其次采用国际领先的基因组编辑技术CRISPR/Cas9构建白木香jaz突变体库；再次采用qRT-PCR和GC-MS技术比较白木香jaz突变体与野生型的沉香倍半萜合成途径酶基因表达及成分合成积累差异；最后筛选高含量沉香倍半萜的白木香jaz突变体，明确AsJAZ对沉香倍半萜合成的调控机制。本项目将为快速敲除控制药用植物不良性状的基因簇和实现遗传改良奠定理论基础，将为高含量药用植物的种质创新和品种优化提供新策略。

沉香属白木香Aquilaria sinensis (Lour.) Gilg为我国特有的珍稀濒危植物，是我国生产名贵芳香类药材沉香的唯一正品植物来源。白木香树体只有受到伤害后才能够在伤口附近产生沉香倍半萜并逐渐积累增多形成沉香药材。项目组推测伤害信号“茉莉酸”途径的抑制因子AsJAZ1阻遏倍半萜合酶基因表达及沉香生成，故提出“AsJAZ作为转录抑制因子阻遏沉香倍半萜基因表达”假说，深入研究发现在拟南芥中过表达白木香AsJAZ1基因会导致拟南芥倍半萜合酶基因TPS21和TPS11基因表达水平显著降低并且对茉莉酸不敏感，故推测茉莉酸途径的抑制因子AsJAZ1阻遏倍半萜合酶基因表达。通过扫描白木香全基因组序列筛选鉴定12个AsJAZ基因家族成员，并分析其启动子包含各种激素相关、组织特异性和转录因子结合元件。转录组结果显示AsJAZ1、AsJAZ2、AsJAZ7/8、AsJAZ9、AsJAZ10和AsJAZ12在通体结香法的结香层中高表表达。实时定量PCR结果显示AsJAZ5、AsJAZ9和AsJAZ10在不同激素处理下的表达变化明显较高。另外，采用白木香胚轴成功诱导并获得的可稳定增殖愈伤组织，并根据AsJAZ4基因设计了单靶标Cas9/sgRNA载体pRGEB32-JAZ4，并利用农杆菌介导的遗传转化体系将CRISPR-Cas9表达盒转入白木香愈伤组织，15个株系检测出Cas9基因，表明植物编辑载体已经转入白木香愈伤组织中。测序结果表明其中7个转基因株系在靶位点发生了基因编辑，编辑效率为63.6%，基因编辑的类型包含碱基缺失和单碱基的插入。高含量沉香倍半萜的白木香jaz突变体的筛选将明确AsJAZ对沉香倍半萜合成的调控机制，也为快速敲除控制药用植物不良性状的基因簇和实现快速遗传改良提供基础数据，也将为高含量药用植物的种质创新和品种改良提供新策略。本项目发表SCI论文3篇，获得发明专利授权2项，荣获世界中医药学会联合会科技进步二等奖1项。