目的 通过分析药用植物长春花Cathranthus roseus萜类吲哚生物碱（terpenoid indole alkaloids，TIAs）合成途径上的抑制转录因子CrZCT1、CrZCT2和CrZCT3间的功能差异，为有效地调控TIAs合成提供必要的理论依据。方法 利用病毒诱导的基因沉默（virus induced gene silencing，VIGS）技术，采用不同的CrZCTs基因沉默组合方式，对CrZCTs进行沉默，分析CrZCTs和TIAs生物合成途径中关键酶基因的表达量以及文多灵和长春质碱的积累量变化。结果 当对CrZCTs进行单个或任意2个沉默时，其他未沉默处理的基因相对表达水平也均会随之下调，且CrZCT1和CrZCT2有相似的表达水平变化。对于7种不同组合的沉默处理下，5个关键酶基因（CrTDC、CrSTR、CrG10H、CrT16H和CrDAT）的相对表达量较对照均有不同程度的上调，为对照的1.43～14.65倍；而文多灵和长春质碱的积累量也均显著提升，分别比对照增加15.27%～128.24%和12.24%～96.35%。结论 由于CrZCTs三者间的同源性和功能的冗余性，基因表达和生物碱的合成均表现出同时沉默3个基因的效果大于任意2个的组合大于1个，且CrZCT1和CrZCT2的作用和能力相似，而CrZCT3促进和调控能力更大。

Objective To provide necessary theoretical basis for effectively regulating synthesis of terpenoid indole alkaloids (TIAs) in medicinal plant Catharanthus roseus, and the functional differences between the inhibitory transcription factors CrZCT1, CrZCT2, and CrZCT3 on the synthetic pathway of TIAs were analyzed in this study. Methods The virus-induced gene silencing (VIGS) technology was used to silence CrZCTs with different gene combinations, the relative expression levels of CrZCTs and five key enzyme genes in the biosynthesis pathway as well as the accumulation levels of vindoline and catharanthine were then analyzed. Results Both mono- and double-silencing of CrZCTs all down-regulated the expression of other unsilenced treated genes when they were silenced, and the CrZCT1 and CrZCT2 had similar expression levels and trends. Compared to the control, the relative expression level of the five key enzyme genes (CrTDC, CrSTR, CrG10H, CrT16H and CrDAT) were all differentially up-regulated among the seven different treatments ranging from 1.43 to 14.65 fold, while the accumulation of vindoline and catharanthine was also significantly elevated, ranging from 15.27%—128.24% and 12.24%—96.35%, respectively. Conclusion The three CrZCTs have similar functions in regulating alkaloid synthesis due to the homology and redundancy among them. Both key enzyme gene expression and alkaloid accumulation promotion effect all exhibited that simultaneous silencing of the all three genes are greater than any two genes greater than one gene, and the effects of CrZCT1 and CrZCT2 are similar, while CrZCT3 has greater promotion and regulation ability.

R286.12

辽宁省教育厅科学研究项目（LJKZ0523）