药学学报, 2018, 53(4): 636-645
何贝轩, 薛英茹, 涂燕华, 高越, 郭美丽. CtCHS4响应茉莉酸甲酯诱导促进了红花醌式查尔酮类化合物的积累[J]. 药学学报, 2018, 53(4): 636-645.
HE Bei-xuan, XUE Ying-ru, TU Yan-hua, GAO Yue, GUO Mei-li. CtCHS4 induces the accumulation of safflower quinone chalcones in response to methyl jasmonate induction[J]. Acta Pharmaceutica Sinica, 2018, 53(4): 636-645.

何贝轩, 薛英茹, 涂燕华, 高越, 郭美丽
第二军医大学药学院, 上海 200433
黄酮类化合物特别是查尔酮类如羟基红花黄色素A、红花红色素等是红花的主要药效成分,研究红花黄酮类的生物合成途径,对于定向调控红花的品质具有重要意义。作为调控黄酮类成分合成的入口酶,查尔酮合酶(CHS)在黄酮类化合物的合成过程中起着重要作用。但到目前为止,CHS在红花黄酮类化合物生物合成过程中的作用尚不十分清楚。茉莉酸甲酯JA/MeJA作为植物信号调节物质可以激活植物体内CHS基因表达。本研究在前期阐明红花的1个CHS基因CtCHS1功能的基础上,作为延续性工作,继续对红花中的其他CHS基因CtCHS2CtCHS4进行研究。应用MeJA刺激花冠,分别于刺激后0、3、6、12 h不同时间点采用qRT-PCR法分析CtCHS2CtCHS4的相对表达量,采用UHPLC/Q-TOF-MS技术分析红花中次生代谢产物的变化。结果表明,CtCHS4的表达量响应MeJA的诱导在3、6 h均明显升高,而CtCHS2的表达量则在诱导后表现出降低的趋势;同时,MeJA诱导后,芦丁、羟基红花黄色素A、D-苯丙氨酸、山柰酚-3-O-β-芸香糖苷、红花红色素的积累量明显提高,特别是羟基红花黄色素A的积累量在诱导后3、6、12 h均显著性的提高(P ≤ 0.05或0.01),而对山柰酚、山柰酚-3-O-β-D-葡萄糖苷、木犀草素、槲皮素-3-β-D-葡糖苷的积累量的影响则不明显。Pearson相关分析结果表明,羟基红花黄色素A、红花红色素的积累量与CtCHS4的表达量均呈显著性正相关(r ≥ 0.8)。提示CtCHS4可能是形成羟基红花黄色素A和红花红色素的关键基因,在红花查尔酮类成分的积累中起着重要作用。CtCHS4-pMAL-C5X重组质粒在BL21(DE3)PlyS原核表达宿主菌中成功表达出CtCHS4活性蛋白,在体外催化底物香豆酰辅酶A和丙二酰辅酶A生成了产物柚皮素。本研究结果进一步完善了红花中CHS基因的功能,为最终阐明红花查尔酮类化合物生物合成途径的关键基因积累了资料。
关键词:    CtCHS4      茉莉酸甲酯      醌式查尔酮      羟基红花黄色素A     
CtCHS4 induces the accumulation of safflower quinone chalcones in response to methyl jasmonate induction
HE Bei-xuan, XUE Ying-ru, TU Yan-hua, GAO Yue, GUO Mei-li
School of Pharmacy, Second Military Medical University, Shanghai 200433, China
Flavonoids, especially chalcones such as hydroxysafflor yellow A and carthamin are the main active ingredients of safflower. To study the biosynthesis pathway of safflower flavonoids is of great significance for the quality control of safflower. Chalcone synthase (CHS) is an enzyme that plays an important role in regulation of the synthesis of flavonoids. However, for the time being, the role of CHS is not yet clear in the biosynthesis of safflower flavonoids. As a plant signaling regulator, JA/MeJA can activate CHS gene expression in plants. CtCHS1, one of the CHS genes in safflower, was elucidated in our previous work. In our continuous search for CtCHSs functions from this plant, other CHS genes CtCHS2 and CtCHS4 in safflower were examined. The floret was stimulated with methyl jasmonate (MeJA) and the transcriptome expression of CtCHS2 and CtCHS4 was analyzed by qRT-PCR at different time points of 0, 3, 6, and 12 h after stimulation. Further metabolites under stimulation by MeJA were analyzed by UHPLC/Q-TOF-MS. The results showed that the expression of CtCHS4 in response to MeJA significantly increased at 3 and 6 h, while the expression of CtCHS2 showed a trend of decrease after induction. Meanwhile, the accumulation of rutin, hydroxysafflor yellow A, D-phenylalanine, kaempferol-3-O-β-rutinoside and carthamin increased obviously. Especially, accumulation of hydroxysafflor yellow A was increased significantly at 3, 6 and 12 h after induction (P ≥ 0.05 or 0.01), but the change in kaempferol, kaempferol-3-O-β-D-glucoside, luteolin, quercetin-3-β-D-glucoside was not significant. The accumulation of hydroxysafflor yellow A and carthamin was positively correlated with the expression abundance of CtCHS4 with Pearson correlation analysis method (r ≥ 0.8). The data suggest that CtCHS4 may be a key gene for forming hydroxysafflor yellow A and carthamin and plays an important role in the accumulation of safflower chalcones. The CtCHS4-pMAL-C5X recombinant vector was successfully expressed in BL21 (DE3) Plys to express the product naringenin in vitro under the catalytic substrates p-coumaryol-COA and malonyl-CoA. The results of this study provide a new insight into synthetic genes involved in flavonoids biosynthetic pathway to elucidate the biosynthesis pathway of safflower chalcones.
Key words:    safflower    CtCHS4    methyl jasmonate    quinonechalcone    hydroxysafflor yellow A   
收稿日期: 2017-11-10
DOI: 10.16438/j.0513-4870.2017-1110
基金项目: 国家自然科学基金资助项目(81473300,81173484);国家"863"项目资助项目(2008AA02Z137).
通讯作者: 高越,Tel:86-10-21-81871302,E-mail:mlguo@126.com;郭美丽,E-mail:gaoyue2000@hotmail.com
Email: mlguo@126.com;gaoyue2000@hotmail.com
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