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青蒿素是从青蒿(Artemisia annua L.)中分离的一种含有过氧桥基团结构的新型倍半萜内酯[1], 对于治疗脑型疟疾和抗氯喹恶性疟疾有特效[2].青蒿素联合治疗法(Artemisinin Combination Therapies, ATCs)是世界卫生组织(WHO)唯一推荐使用的基于青蒿素治疗疟疾的最有效的治疗方法[2].青蒿中青蒿素含量很低, 从天然青蒿中提取青蒿素远不能满足巨大市场需求[3], 提高青蒿素产量成为一项迫切任务.目前, 通过代谢工程提高青蒿素产量主要在两方面开展研究:采用合成生物学在酵母中合成青蒿酸并进而光氧化形成青蒿素[4]; 采用代谢工程技术培育青蒿素超高产青蒿新品种[5]. Keasling团队[6]在酵母中成功的实现了青蒿素的部分合成, 但是其现有的年产量有限且成本较高, 无法满足巨大市场需求.培育青蒿素超高产青蒿新品种仍然是很有前景的研究方向.开展青蒿素合成途径分子生物学研究是实现青蒿代谢工程育种的前体.甲瓦龙酸途径(MVA途径)为青蒿素生物合成提供基本5碳前体, 3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl-CoA reductase, HMGR)催化3-羟基-3甲基戊二酰辅酶A形成甲羟戊酸.在植物中, HMGR基因以家族形式存在, 提高HMGR表达能促进萜类积累[7].在青蒿中超量表达外源HMGR基因能显著提高青蒿素含量[8].然而, 关于青蒿HMGR家族研究未见报道, 各家族成员在青蒿素生物合成中的具体贡献也不清楚.
本研究以青蒿为研究对象, 根据我们和上海交通大学联合测定的青蒿全基因组序列(未公开), 结合RACE技术克隆青蒿HMGR家族基因, 并对其进行了生物信息学分析.通过实时荧光定量PCR技术, 对3条HMGR基因进行了组织表达特异性分析, 以及MeJA和损伤诱导的表达模式分析.通过不同层面的实验数据以及分析, 解析青蒿HMGR家族成员不同功能.
Molecular Cloning and Expression Analysis of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) Reductase Gene Family Upon MeJA and Mechanical Injury Treatments in Artemisia annua L.
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摘要: 通过全基因组序列测定和RACE技术, 从青蒿(Artemisia annua L.)克隆出了3条编码HMGR的cDNA序列:AaHMGR1, AaHMGR2和AaHMGR3, 并对3条基因进行生物信息学分析.结果表明:3条序列与其他物种的HMGR具有高度相似性但基因组结构不一致; 组织表达分析中, AaHMGR1在茎中表达量最高, 而AaHMGR2和AaHMGR3在花中表达量较高; 外源茉莉酸甲酯(MeJA)诱导处理后AaHMGR1的表达在0.5 h时达到最大值, AaHMGR2和AaHMGR3则轻微响应; 机械损伤3 h后能够使AaHMGR1的表达量上调约700倍, 而AaHMGR2, AaHMGR3上调幅度却不大.综上研究结果表明, AaHMGR1基因可能在青蒿素的生物合成中起着更为重要的作用.Abstract: Three full-length cDNA of HMGR, i.e. AaHMGR1, AaHMGR2 and AaHMGR3, were cloned from Artemisia annua. Bioinformatic and phylogenetic analyses revealed that all the three AaHMGRs shared extensive homology with other plant species and belonged to the plant HMGR family; however, the corresponding coding region of the genome structure was different. The expression of AaHMGR1 was the highest in the stem, while the expression of AaHMGR2 and AaHMGR3 was relatively high in the flower. The expression of AaHMGR1 reached the maximum at 0.5 h after MeJA induction, and then decreased gradually, while AaHMGR2 and AaHMGR3 responded but slightly. The expression of AaHMGR1 increased by about 700 times at 3 h after mechanical injury, while that of AaHMGR2 and AaHMGR3 was not significant. The above results suggested that AaHMGR1 may play a crucial role in artemisinin biosynthesis.
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Key words:
- Artemisia annua /
- HMGR /
- MeJA /
- wound /
- artemisinin .
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表 1 基因克隆相关引物
引物名称 类型 引物序列(5'-3') HMGR1-3 3'-RACE 1-ACTGGTGATAATGATGATGGTA 2-ACAGATCAAGCAGAGACATGTC HMGR2-3 3'-RACE 1-CAGTCACCATGCCTTCAATC 2-CAACTACTTCTGTTCCTCTCC HMGR3-3 3'-RACE 1-TAGATTGTGAGTCAGATGTGGTC 2-TCTTGATGGTTTGCCTCTAGAG HMGR1-5 5'-RACE 1-GATCTTGTCGCGCCATCTGTGA 2-GTAATGGTAATGCGTCTGAGGCT HMGR2-5 5'-RACE 1-CGAGTTTCGCCATTCATGGATGT 2-CGAGTTTCGCCATTCATGGATGT HMGR3-5 5'-RACE 1-CCAAGCTTGGATTCCAACGAGT 2-CCACAAGAGCTCACACGACTGT M13 3'-RACE GTTTTCCCAGTCACGAC UPM/NUP 5'-RACE U-CTAATACGACTCACTATAGGGC N-AAGCAGTGGTATCAACGCAGAG 表 2 基因表达分析引物
引物名称 引物序列(5'-3')F 引物序列(5'-3')R HMGR1RT ACTGGTGATAATGATGATGGTA TATCTTAGCCGCTCTATAACAA HMGR2 RT CAATCTATCATCCGTTCTTC AGAGATAATGTGAGGAATCA HMGR3RT ATACAACCGCTCCACCAG ACCAGAAGAAGAACAAGAACAC EF1α GACCTACTCTCCTTGAAG GTTCCAATACCACCAATC 表 3 HMGR生物信息学分析
蛋白 分子量 等电点pI HMG-CoA结合基序 NADP(H)结合基序 AaHMGR1 61.1 kDa 6.52 AaHMGR2 62.9 kDa 6.58 EMPIGYVQIP TTEGCLVA DAMGMNM GTVGGGT AaHMGR3 62.4 kDa 6.08 -
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