Molecular Cloning and Expression Analysis of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) Reductase Gene Family Upon MeJA and Mechanical Injury Treatments in <i>Artemisia annua</i> L.

Yu-pei CHEN; Tian SHEN; Fei QIU; Min CHEN; Zhi-hua LIAO; Xiao-zhong LAN; Chun-xian YANG

doi:10.13718/j.cnki.xdzk.2017.12.007

2017 Volume 39 Issue 12

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Yu-pei CHEN, Tian SHEN, Fei QIU, et al. 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.[J]. Journal of Southwest University Natural Science Edition, 2017, 39(12): 44-51. doi: 10.13718/j.cnki.xdzk.2017.12.007

Citation:

Yu-pei CHEN, Tian SHEN, Fei QIU, et al. 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.[J]. Journal of Southwest University Natural Science Edition, 2017, 39(12): 44-51. doi: 10.13718/j.cnki.xdzk.2017.12.007

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.

1.
Key Laboratory of the Ministry of education of the ecological environment of the Three Gorges Reservoir Area/ Chongqing Engineering and Technology Research Center for Sweet potato, School of Life Sciences, Southwest University, Chongqing 400715, China
2.
Jiangchuan No.1 Middle School, Yuxi Yunnan 652600, China
3.
School of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
4.
School of Food Sciences, Xizang Agricultural and Husbandry College, Linzhi, Tibet 86000, China

More Information

Corresponding author: Chun-xian YANG
Received Date: 27/12/2016
Available Online: 20/12/2017
MSC: Q946.889

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.
- Artemisia annua,
- HMGR,
- MeJA,
- wound,
- artemisinin

References

[1]	国家药典委员会.中华人民共和国药典[M].北京:中国医药科技出版社, 2010. Google Scholar
[2]	FAUTH, MOHANTY S. Guidelines for the Treatment of Malaria [J]. World Health Organization, 2006, 6(2):632-646. Google Scholar
[3]	WANG Y Y, JING F Y, YU S Y, et al. Co-Overexpression of the HMGR and FPS Genes Enhances Artemisinin Content in Artemisia annua L. [J]. J Med Plant Res, 2011, 5(15):3396-3403. Google Scholar
[4]	HALE V, KEASLING J D, RENNINGER N, et al. Microbially Derived Artemisinin:A Biotechnology Solution to the Global Problem of Access to Affordable Antimalarial Drugs [J]. Am J Trop Med Hyg, 2007, 77(6 Suppl):198-202. Google Scholar
[5]	LIU B, WANG H, DU Z, et al. Metabolic Engineering of Artemisinin Biosynthesis in Artemisia annua L. [J]. Plant Cell Rep, 2011, 30(5):689-694. doi: 10.1007/s00299-010-0967-9 CrossRef Google Scholar
[6]	RO D K, PARADISE E M, OUELLET M, et al. Production of the Antimalarial Drug Precursor Artemisinic Acid in Engineered Yeast [J]. Nature, 2006, 440(7086):940-943. doi: 10.1038/nature04640 CrossRef Google Scholar
[7]	HARKER M, HOLMBERG N, CLAYTON J C, et al. Enhancement of Seed Phytosterol Levels by Expression of an N-Terminal Truncated Hevea Brasiliensis (Rubber tree) 3-Hydroxy-3-Methylglutaryl-CoA Reductase [J]. Plant Biotechnol J, 2003, 1(2):113-121. doi: 10.1046/j.1467-7652.2003.00011.x CrossRef Google Scholar
[8]	AQUIL S, HUSAINI A M, ABDIN M Z, et al. Overexpression of the HMG-CoA Reductase Gene Leads to Enhanced Artemisinin Biosynthesis in Transgenic Artemisia annua Plants [J]. Planta Med, 2009, 75(13):1453-1458. doi: 10.1055/s-0029-1185775 CrossRef Google Scholar
[9]	LIAO Z H, TAN Q M, CHAI Y R, et al. Cloning and Characterisation of the Gene Encoding HMG-CoA Reductase from Taxus Media and Its Functional Identification in Yeast [J]. Functional Plant Biology, 2004, 31(1):785-801. Google Scholar
[10]	XIANG L E, ZHU S Q, ZHAO T F, et al. Enhancement of Artemisinin Content and Relative Expression of Genes of Artemisinin Biosynthesis in Artemisia annua by Exogenous MeJA Treatment [J]. Plant Growth Regulation:2014, 75(2):435-441. Google Scholar
[11]	BOTER M. Conserved MYC Transcription Factors Play a Key Role in Jasmonate Signaling Both in Tomato and Arabidopsis [J]. Genes Dev, 2004, 18(13):1577-1591. doi: 10.1101/gad.297704 CrossRef Google Scholar
[12]	WANG H H, MA C F, LI Z Q, et al. Effects of Exogenous Methyl Jasmonate on Artemisinin Biosynthesis and Secondary Metabolites in Artemisia annua L. [J]. Industrial Crops and Products, 2010, 31(2):214-218. doi: 10.1016/j.indcrop.2009.10.008 CrossRef Google Scholar
[13]	LIU D H, ZHANG L D, LI C X, et al. Effect of Wounding on Gene Expression Involved in Artemisinin Biosynthesis and Artemisinin Production in Artemisia annua [J]. Russ J Plant Physiol, 2010, 57(6):882-886. doi: 10.1134/S102144371006018X CrossRef Google Scholar

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沈阳化工大学材料科学与工程学院沈阳 110142

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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.

1. 材料与方法

1.1. 植物材料以及处理

材料为具有相同遗传背景青蒿快繁幼苗(Xiang et al)^[10].参照Wang等^[12]和Liu等^[13]的方法进行MeJA和损伤处理.将实验材料用液氮研磨, 提取RNA后反转录合成cDNA用于后续实验, 提取RNA和反转录方法参考Liu^[13]等的方法.

1.2. 基因克隆

根据青蒿全基因组、trichOME Database、NCBI上已经报道的青蒿HMGR基因片段, 利用Vector NTI suite 11.5软件进行拼接与比对找到3条不同的青蒿HMGR序列, 根据这3条序列分别设计3'RACE巢式PCR引物(表 1), 利用引物、dNTP和HiFi酶等按巢式PCR程序扩增获得AaHMGRs的3'RACE, 利用拼接结果设计5'RACE巢式PCR引物(表 1), 按照同样程序获得AaHMGRs的5'RACE, 并拼接获得AaHMGRs的电子全长, 用PCR扩增获得AaHMGRs的物理全长.

1.3. 生物信息学分析

通过Vector NTI suite 11.5软件进行CDS的查找和翻译, 在NCBI网站的BLAST进行核酸和氨基酸序列比对; 用ClustalX软件进行氨基酸序列多重比对, 用MEGA4.1的Neighbor-joining进行进化树的构建.蛋白质的基本性质在ExPASy网站在线分析.

1.4. 表达分析

根据获得的AaHMGRs利用Beacon designer软件设计荧光定量特异性引物(表 2), EF1α为内参基因, 用染料iTaq^TM Universal SYBR^® Green Supermix通过iQ5 cycler荧光定量PCR仪反应并根据2^-ΔΔCt方法计算得到AaHMGRs的相对表达量, 反应的条件为:预熔解95 ℃ 2 min, 熔解95 ℃ 15 s, 退火58.2 ℃ 20 s, 72 ℃延伸20 s, 循环40次, 熔解曲线, 55~95 ℃, 80个循环, 每循环升温0.5 ℃(Liu et al).

3. 讨论

本研究获得的AaHMGR1, AaHMGR2, AaHMGR3与其它已经报道的植物HMGR具有高度同源性, 且均包含有HMGR所特有的HMG-CoA和NADP(H)保守结构域^[9]. 3条HMGR的DNA结构不相同, 且在组织中表达中AaHMGR1在茎中表达最高, 而AaHMGR2和AaHMGR3在花中表达最高, 说明AaHMGR2和AaHMGR3的本底水平表达比AaHMGR1高, 更近一步说明可能在激素或损伤诱导时AaHMGR1更有利于青蒿素生物合成.实验结果显示在MeJA和损伤处理后AaHMGRs基因的响应模式确有统计学差异, AaHMGRs基因受MeJA和损伤诱导, 特别是受损伤诱导, 与先前的研究结果一致:Liu等^[13]用损伤处理青蒿后检测HMGR(AF142473) 的表达量及青蒿素含量, 在处理后HMGR基因表达量在2h达到最大值, 青蒿素含量在2h后显著提高并在4h时达到最大值.可利用损伤青蒿作为提高青蒿素的有效方法之一. MeJA在短时间处理后, AaHMGR1的表达量显著提高, 结果与Xiang^[10]的研究结果相一致, 可考虑尝试利用AaHMGR1来提高青蒿素的生物合成.青蒿素的含量在MeJA处理后也得到显著提高, 这可能是MeJA的处理, 激活了参与信号转导同时参与青蒿素合成的转录因子的表达, 从而进一步引起下游参与青蒿素合成的基因的表达, 最终导致青蒿素含量的提高.目前研究发现很多转录因子均参与MeJA的信号转导, 并启动下游基因的表达^[11].损伤处理后, AaHMGR1的表达量和青蒿素的含量显著提高, 这可能是处理后, 诱导了参与损伤信号转导同时也参与青蒿素合成的转录因子表达, 从而进一步引起下游参与青蒿素合成的基因的表达, 最终导致青蒿素含量的提高.但是关于损伤所涉及的转录因子的研究还较少, 因此这方面的研究还需要再深入, 探寻其提高青蒿素含量的分子机制.

Figure (6) Table (3) Reference (13)

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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.