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中药红花(Carthami Flos)是菊科植物红花(Carthamus tinctorius L.)的干燥花,传统本草学著作《本草纲目》记载,红花具有活血散瘀,通经止痛的功效[1],其药材和制剂在临床上被广泛用于心脑血管疾病的预防和治疗。现代药理研究表明,其主要药效物质是以羟基红花黄色素A(hydroxysafflower yellow A,HSYA)为代表的查尔酮类化合物和以菸花苷为代表的黄酮醇类化合物,这些化合物均具有良好的心脑血管损伤保护活性[2-3]。红花药材的产量偏低,每平方千米产量仅为18.0~22.5 t[4],其中特有的HSYA[5]、红花红色素等查尔酮类成分在不同品种间差异较大[6]。由于红花中的查尔酮类成分仅特异性地存在于花冠中[7],加之体外组织培养再生率低[8]等原因,对其功能基因的研究工作一直进展缓慢。特别是对于HSYA等红花特有的有效成分,其生物合成相关的功能基因尚不完全清楚,合成通路也未被完全解析[9]。因此,用现代分子生物学技术手段以提高药效物质的含量,是提高红花品质,节约土地资源、降低制药成本的一条新途径。
短链脱氢酶/还原酶(short-chain dehydrogenases/reductases,SDR)在植物次生代谢物的生物合成中广泛参与各类碳-氧双键,碳-碳双键以及烯酮键的氧化还原催化反应。根据SDRs基因序列的特征结构,SDRs超家族可以被分为5个亚家族[10-14]。最早发现并且进行鉴定的两类主要短链还原酶命名为classical和extend,classical类的SDRs基因拥有长度约为250个氨基酸残基,被称为Extended类的SDRs基因在碳基末端因其含有多余的约100个氨基酸残基而得名。另外3种类型SDRs基因分别被命名为intermediate、complex和divergent。这些类型的SDRs基因基于其结合辅酶类型和结合催化位点的不同进行命名分类。此外,SDRs存在与传统类型不同的含有“rossmann-fold”保守结构域的氧化还原酶结构[15-18]。
黄酮类化合物起源于莽草酸途径和苯丙素生物合成途径,1个香豆酰辅酶A(coumaroyl CoA)和3个丙二酰辅酶A(malonyl CoA)在查尔酮合酶的作用下生成二氢查尔酮,然后经查尔酮异构酶催化为二氢黄酮,进一步在各类还原酶,聚合酶和糖基转移酶的作用下,生成终端次生代谢产物组合[19-21]。红花中所含的主要有效成分HSYA具有查尔酮式结构,本课题组前期研究认为:HSYA从前体物质到合成,中间存在必不可少的氧化还原过程。短链脱氢还原酶家族广泛参与植物体内次生代谢,这一类还原酶都带有相似的折叠结构以及催化位点,已有研究表明,其对苯丙烷代谢途径起重要作用[22-23],但有关红花中还原酶基因相关报道较少[24]。故笔者通过对红花转录组数据库、基因表达谱数据库以及代谢组数据库进行分析,筛选在HSYA生物合成途径的关键还原酶基因,并进行功能验证,以期揭示红花次生代谢成分生物合成途径,为定向调控红花的品质提供科学依据。
Characterization and function of short-chain dehydrogenases/reductases in hydroxysafflower yellow A biosynthesis pathway
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摘要:
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T2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T2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T2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。 Abstract: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 T2 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 T2 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. -
Key words:
- short-chain dehydrogenase reductase /
- hydroxysafflor yellow A /
- safflower
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图 6 真核表达载体构建及阳性鉴定电泳图
注:1. CtSDR3基因开放阅读框(ORF)区扩增产物电泳图,a、b泳道均为CtSDR3基因ORF区克隆PCR产物;2. 真核表达载体pMT-39载体酶切产物电泳图,a、b泳道为CtSDR3 PCR产物,c泳道为pMT-39载体,d、e泳道为pMT-39线性化载体;3. pMT39-CtSDR3重组载体阳性转化子鉴定电泳图,a、b泳道为阳性转化子菌液PCR产物;4. pMT39-CtSDR3质粒转化农杆菌GV3101,a、c和e泳道为空白对照组,b、d和f泳道为阳性克隆菌液PCR产物;5. 红花pMT39-CtSDR3阳性转化植株鉴定PCR产物电泳图,1~19为待鉴定植株,p为pMT39-CtSDR3质粒,k为空白组,WT为野生型红花植株
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