羟基红花黄色素A生物合成途径短链还原酶基因的特征及功能研究

Characterization and function of short-chain dehydrogenases/reductases in hydroxysafflower yellow A biosynthesis pathway

目的探究参与红花黄酮类生物合成途径特别是羟基红花黄色素A(HSYA)关键短链脱氢还原酶基因(SDRs)的功能。方法基于红花转录组数据库以及代谢组数据库,筛选参与HSYA生物合成途径的SDRs,用qRT-PCR法分析表达模式。采用无缝克隆技术构建过表达载体,以农杆菌GV3101介导遗传转化云南巍山红花品系,对转基因T_(2)代植株进行阳性验证,并对花冠SDRs的基因表达量进行分析,UPLC-Q-TOF/MS法测定次生代谢物的含量。结果筛选出3个参与HSYA生物合成途径的关键短链脱氢还原酶基因CtSDR1、CtSDR2、CtSDR3,表达量从高到低依次为花冠>叶>茎>根。花冠中表达量随花冠发育逐渐升高。对转基因T_(2)代植株进行阳性验证后的花冠进行qRT-PCR分析发现:与空白对照组相比,转CtSDR3过表达T_(2)代阳性植株花冠中CtSDR3基因的转录水平增加了2~3倍,次生代谢物HSYA的含量提高了7.1%~16.6%(P<0.05)。结论CtSDR3可能参与了红花中黄酮类化合物特别是HSYA的生物合成,为阐释CtSDR3在HSYA生物合成途径中的功能提供了数据支撑。

Objective To explore the function of short-chain dehydrogenases/reductases(SDRs)in safflower flavonoid,especially hydroxysafflower yellow A(HSYA)biosynthesis.Methods SDRs involved in HSYA biosynthesis pathway were screened based on safflower transcriptome database and metabolome database.The expression pattern was analyzed by qRT-PCR.The overexpression vector was constructed by seamless cloning technology,then genetically transformed to the Yunnan Weishan safflower strain by Agrobacterium gv3101.The transgenic T_(2) generation plants were positively verified,and the gene expression of corolla SDRs was analyzed.The content of secondary metabolites was assayed by UPLC-Q-TOF/MS.Results Three SDRs genes named CtSDR1,CtSDR2 and CtSDR3 involved in HSYA biosynthesis pathway were screened.Their expression in safflower from high to low was corolla>leaf>stem>root.The expression level in corolla increased gradually with corolla development.qRT-PCR analysis of corolla with positive verification of genome insertion sequence showed that the transcription level of CtSDR3 in corolla of T_(2) positive plants increased by 2~3 times compared with the blank control group,and the content of secondary metabolite HSYA increased by 7.1%~16.6%(P<0.05).Conclusion CtSDR3 may be involved in the biosynthesis of flavonoids,especially HSYA,in safflower.It provides the support data for explaining the function of CtSDR3 in HSYA biosynthesis pathway.