铜胁迫对茅苍术3种药效成分积累及其生物合成2种关键酶基因表达的影响
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摘要
目的研究铜胁迫对茅苍术药效成分β-桉叶醇、苍术酮和苍术素积累及其生物合成关键酶3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGR)、法呢基焦磷酸合酶(FPPS)基因表达的影响。方法在铜胁迫下,采用实时荧光定量PCR(qRT-PCR)法测定茅苍术HMGR、FPPS的表达量;采用高效液相色谱法测定茅苍术3种药效成分的含量;采用SPSS软件对两者进行相关性分析,DPS软件进行灰色关联度分析。结果当铜胁迫在100 mg/kg以内时,茅苍术根茎内FPPS表达量与苍术酮含量略有增加;但铜浓度继续增加时,HMGR、FPPS表达量与3种药效成分含量呈不同程度的下降趋势。铜胁迫下HMGR、FPPS的表达量与β-桉叶醇、苍术酮和苍术素的含量呈显著正相关(P<0.05)。灰色关联度分析发现铜胁迫下茅苍术根茎中β-桉叶醇、苍术酮的含量与HMGR、FPPS的表达量关联较大,FPPS基因的表达对β-桉叶醇和苍术酮合成贡献较大,但苍术素含量与这2种关键酶基因的表达关联度较小。结论阐明了铜胁迫下茅苍术2种关键酶基因表达量与3种药效成分含量的变化规律,揭示了铜胁迫下茅苍术药效成分β-桉叶醇、苍术酮与萜类生物合成关键酶HMGR、FPPS的关系,有助于进一步深入研究铜胁迫下茅苍术药效成分合成的分子调控机制,为提高茅苍术药材的品质提供理论基础。
Objective To explore the effect on the accumulation of medicinal compositions β-eudesmol, atractylon, atractylodin and key enzyme genes 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR) and farnesyl pyrophosphate synthase(FPPS) expression in biosynthesis of Atractylodes lancea under copper stress. Methods Under copper stress, the expression of key enzyme genes HMGR and FPPS in A. lancea was determined by real-time fluorescence quantitative PCR; the content of three medicinal components in A. lancea were determined by HPLC; The correlation analysis was performed with SPSS, and DPS software for grey correlation analysis. Results When the copper stress concentration was within 100 mg/kg, the expression of FPPS and the content of atractylon in the rhizomes of A. lancea increased slightly. However, when the copper concentration continued to increase, the expression levels of HMGR and FPPS and three medicinal components content of A. lancea showed a different degrees of downward trend. The expression levels of HMGR and FPPS were positively correlated with the content of β-eudesmol, atractylon, and atractylodin(P < 0.05) under copper stress. Grey relational analysis showed that the content of β-eudesmol and atractylon in the rhizomes was significantly correlated with the expression of HMGR and FPPS of A. lancea under copper stress. The expression of FPPS gene had the larger contribution on the composition of β-eudesmol and atractylon. However, the correlation between the content of atractylodin and the expression of these two key enzyme genes was relatively small. Conclusion This study clarified the change regulation of two key enzyme gene expression and the content of three medicinal compositions, and revealed the relationship between β-eudesmol, atractylon and HMGR and FPPS, the key enzymes in terpene biosynthesis of A. lancea under copper stress. It contributed to the further study of the molecular regulation mechanism of the synthesis of medicinal constituents under copper stress and provided a theoretical basis for improving the quality of A. lancea.
Objective To explore the effect on the accumulation of medicinal compositions β-eudesmol, atractylon, atractylodin and key enzyme genes 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR) and farnesyl pyrophosphate synthase(FPPS) expression in biosynthesis of Atractylodes lancea under copper stress. Methods Under copper stress, the expression of key enzyme genes HMGR and FPPS in A. lancea was determined by real-time fluorescence quantitative PCR; the content of three medicinal components in A. lancea were determined by HPLC; The correlation analysis was performed with SPSS, and DPS software for grey correlation analysis. Results When the copper stress concentration was within 100 mg/kg, the expression of FPPS and the content of atractylon in the rhizomes of A. lancea increased slightly. However, when the copper concentration continued to increase, the expression levels of HMGR and FPPS and three medicinal components content of A. lancea showed a different degrees of downward trend. The expression levels of HMGR and FPPS were positively correlated with the content of β-eudesmol, atractylon, and atractylodin(P < 0.05) under copper stress. Grey relational analysis showed that the content of β-eudesmol and atractylon in the rhizomes was significantly correlated with the expression of HMGR and FPPS of A. lancea under copper stress. The expression of FPPS gene had the larger contribution on the composition of β-eudesmol and atractylon. However, the correlation between the content of atractylodin and the expression of these two key enzyme genes was relatively small. Conclusion This study clarified the change regulation of two key enzyme gene expression and the content of three medicinal compositions, and revealed the relationship between β-eudesmol, atractylon and HMGR and FPPS, the key enzymes in terpene biosynthesis of A. lancea under copper stress. It contributed to the further study of the molecular regulation mechanism of the synthesis of medicinal constituents under copper stress and provided a theoretical basis for improving the quality of A. lancea.
引文
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[15]贾鹏,王媛,陈涛,等.苦荞MT2基因的克隆及其对铜胁迫的响应[J].农业生物技术学报,2017,25(6):874-883.
[16]王川,李昆伟,魏宇昆,等.Cu2+胁迫对丹参生长及有效成分积累的影响[J].植物研究,2012,32(1):124-128.
[17]魏麟,黎晓英,刘胜贵,等.鱼腥草HMGR基因cDNA克隆、差异表达及蛋白质结构分析[J].中草药,2017,48(18):3815-3819.
[2]戴红君,程金花,虞德容,等.中药茅苍术研究进展[J].江苏农业科学,2016,44(11):26-28.
[3]吴佳新.茅苍术挥发油成分及药理活性综述[J].江苏农业科学,2016,44(3):28-30.
[4]Yin M,Xiao C C,Chen Y,et al.A new sesquiterpenoid glycoside from rhizomes of Atractylodes lancea[J].Chin Herb Med,2015,7(4):371-374.
[5]蒋玲,谷巍,巢建国,等.濒危药用植物茅苍术法呢基焦磷酸合酶基因克隆及其表达分析[J].中草药,2017,48(4):760-766.
[6]Bhambhani S,Lakhwani D,Shukla T,et al.Genes encoding members of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR)gene family from Azadirachta indica and correlation with azadirachtin biosynthesis[J].Acta Physiol Plant,2017,39(2):65.
[7]Qian J,Liu Y,Chao N,et al.Positive selection and functional divergenceof farnesyl pyrophosphate synthase genes in plants[J].BMC Molecular Biol,2017,18(1):3.
[8]Liu X M,Tao T T,Meng X X,et al.Cloning and expression analysis of a farnesyl diphosphate synthase(FPPS)gene from Chamaemelum nobile[J].Not Bot Horti Agrobot Cluj Napoca,2017,45(2):358-364.
[9]陈诚,林晨.苏南地区耕地质量评价与分区保护研究[J].长江流域资源与环境,2016,25(12):1860-1869.
[10]赵胡,唐俊,郑文教.重金属Cu2+胁迫对红树植物秋茄幼苗生长及某些生理特性的影响[J].海洋科学,2016,40(4):65-72.
[11]桑晓华,谷巍,巢建国,等.茅苍术内参基因筛选及其在活性成分生物合成研究中的应用[J].植物生理学报,2017,53(9):1680-1686.
[12]杨林林,张涛,杨利民,等.生态因子对人参皂苷合成及其关键酶基因表达的影响[J].中草药,2017,48(20):4296-4305.
[13]王黎,罗静,裴瑾,等.橘核柠檬苦素类化合物积累与功能基因表达的灰色关联度分析[J].中草药,2017,48(2):345-350.
[14]Moravcova S,Tuma J,Ducaiova Z K,et al.Influence of salicylic acid pretreatment on seeds germination and some defence mechanisms of Zea mays plants under copper stress[J].Plant Physiol Bioch,2018,122:19-30.
[15]贾鹏,王媛,陈涛,等.苦荞MT2基因的克隆及其对铜胁迫的响应[J].农业生物技术学报,2017,25(6):874-883.
[16]王川,李昆伟,魏宇昆,等.Cu2+胁迫对丹参生长及有效成分积累的影响[J].植物研究,2012,32(1):124-128.
[17]魏麟,黎晓英,刘胜贵,等.鱼腥草HMGR基因cDNA克隆、差异表达及蛋白质结构分析[J].中草药,2017,48(18):3815-3819.
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