遮阴对珠子参皂苷合成关键酶基因表达和皂苷积累的影响
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摘要
为研究不同遮阴处理下皂苷合成关键酶表达对珠子参总皂苷合成与积累的影响,以全光照、30%遮阴和50%遮阴3种处理下的盆栽珠子参为材料,分别用实时荧光定量PCR法和紫外分光光度法测定了不同生长时期珠子参叶和根茎中3个关键酶基因(CAS,DS,β-AS)的表达和总皂苷含量。结果表明,在开花期CAS,DS,β-AS均在珠子参地上部分高表达,30%遮阴处理显著抑制了叶中CAS的表达而促进了茎,叶和花中DS,β-AS的表达,推测在此时期皂苷合成的主要部位为叶。整个生长周期内,DS,β-AS的表达与叶中总皂苷含量的变化均呈现出"低—高—低"的趋势,相关系数分析表明,两者之间为正相关关系。遮阴处理下DS,β-AS的总体表达水平和总皂苷含量远高于全光照处理,其中以30%遮阴处理对总皂苷积累的促进作用最明显。因此建议在人工栽培珠子参时,采取30%的遮阴处理,以利于珠子参皂苷的积累和品质的提升。
To explore the effects of shading and the expression of key enzyme genes on the synthesis and accumulation of Panax japonicus var. major saponins, different shading treatments(0%, 30%,50%) of potted P. japonicus var. major were used as test materials, the expression of three key enzyme genes(CAS,DS,β-AS)of leaves and rhizomes in different growth periods of P. japonicus var. major was determined by real-time quantitative PCR, the content of total saponins was determined by ultraviolet spectrophotometry. The results indicated that, in flowering stage, CAS,DS,β-AS were highly expressed in the aerial parts of P. japonicus var. major, 30% shading treatment significantly inhibited the expression of CAS in leaves and promoted the expression of DS and β-AS in stems, leaves and flowers, it was speculated that the main part of saponin synthesis was leaf in this stage. Both the expression levels of DS and β-AS and changes in the content of total saponins in leaves showed a tendency of low-high-low throughout the growth cycle, correlation coefficient analysis showed that there was a positive correlation between them. Compared with control, the expression levels of DS and β-AS and the content of total saponins were greatly enhanced under shading treatment, 30% shading treatment significantly promoted the accumulation of total saponins. Therefore, it is suggested that 30% shading treatment should be applied to the artificial cultivation of P. japonicus var. major, which is beneficial to the accumulation and quality improvement of saponins.
To explore the effects of shading and the expression of key enzyme genes on the synthesis and accumulation of Panax japonicus var. major saponins, different shading treatments(0%, 30%,50%) of potted P. japonicus var. major were used as test materials, the expression of three key enzyme genes(CAS,DS,β-AS)of leaves and rhizomes in different growth periods of P. japonicus var. major was determined by real-time quantitative PCR, the content of total saponins was determined by ultraviolet spectrophotometry. The results indicated that, in flowering stage, CAS,DS,β-AS were highly expressed in the aerial parts of P. japonicus var. major, 30% shading treatment significantly inhibited the expression of CAS in leaves and promoted the expression of DS and β-AS in stems, leaves and flowers, it was speculated that the main part of saponin synthesis was leaf in this stage. Both the expression levels of DS and β-AS and changes in the content of total saponins in leaves showed a tendency of low-high-low throughout the growth cycle, correlation coefficient analysis showed that there was a positive correlation between them. Compared with control, the expression levels of DS and β-AS and the content of total saponins were greatly enhanced under shading treatment, 30% shading treatment significantly promoted the accumulation of total saponins. Therefore, it is suggested that 30% shading treatment should be applied to the artificial cultivation of P. japonicus var. major, which is beneficial to the accumulation and quality improvement of saponins.
引文
[1] 中国药典.一部[S]. 2015
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[3] 王小平,白吉庆,王亚恒,等. 配伍对珠子参中4个皂苷类成分药代动力学的影响[J]. 中国实验方剂学杂志,2017,23(6):111.
[4] 刘爱华, 石孟琼, 杨文雁, 等. 珠子参总皂苷对大鼠心肌缺血/再灌注损伤的保护作用及机制研[J].中国临床药理学与治疗学, 2013, 18(11):1224.
[5] 宋小妹,李林,杨光明.不同产地珠子参中竹节皂苷IVa含量测定[J].中国中药杂志,2010,35(7):885.
[6] 宁张弛,刘振丽,宋志前. HPLC-QqQ-MS测定珠子参中5种皂苷类成分的含量[J]. 中国中药杂志,2016, 41(5):874.
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[8] 张绍鹏, 金健, 胡炳雄, 等. 珍稀药用植物珠子参的转录组测序及分析[J]. 中国中药杂志, 2015, 40(11):2084.
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[11] 罗志勇, 陆秋恒, 刘水平, 等. 人参植物皂苷生物合成相关新基因的筛选与鉴定[J]. 生物化学与生物物理学报, 2003, 35(6): 554.
[12] 吴亚运, 赵小龙, 陈平, 等. 珠子参β-香树素合成酶基因的克隆和生物信息学分析[J]. 生物技术通报, 2017, 33(2):109.
[13] 黄文静,谢培,杨洁, 等. 珠子参皂苷合成途径三个关键酶基因CAS、DS和β-AS时空表达分析[J]. 中国农学通报, 2017, 34(7):31.
[14] 赵新礼. 珠子参生态学和生物学特性的研究[J]. 现代中药研究与实践, 2015, 29(6):14.
[15] 赵仁, 赵毅, 何双凌, 等. 珠子参生产技术标准操作规程(SOP)[J].中国现代中药, 2014, 16(7): 556.
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[17] 李凤英, 王英姿, 王晴, 等. 正交试验法优化珠子参总皂苷提取工艺[J]. 北京中医药大学学报, 2016,36(2):151.
[18] Haralampidis K, Trojanowska M, Osbourn A E. Biosynthesis of triterpenoid saponins in plants[J]. Adv Biochem Eng Biot,2002, 75:31.
[19] 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.
[20] 杨金玲, 高丽丽, 朱平. 人参皂苷生物合成研究进展[J]. 药学学报, 2013, 48(2):170.
[21] 明乾良,韩婷,黄芳, 等. 人参皂苷生物合成途径及其相关酶的研究进展[J]. 中草药, 2010, 41(11): 1913.
[22] 吴琼, 周应群, 孙超, 等. 人参皂苷生物合成和次生代谢工程[J]. 中国生物工程杂志, 2009,29(10):102.
[23] 黄文静, 孙晓春, 王楠, 等. 遮阴对珠子参生长发育及光合生理特性的影响[J]. 中国农学通报, 2017, 34(5):49.
[24] 黄文静, 王楠, 李铂, 等. 不同栽培年限珠子参在不同生长期的光合特性及保护酶活性研究[J]. 中国现代中药, 2017,19(10):1415.
[25] 徐克章, 张美善, 武志海, 等. 人参不同生育期叶片光合作用变化的研究[J]. 作物学报, 2006, 32(10): 1519.
[26] 张治安, 徐克章, 任跃英, 等. 光照条件对参株碳水化合物和人参皂甙含量的影响[J]. 吉林农业大学学报,1994, 16(3):15.
[2] 刘万里, 刘婷, 何忠军, 等. 珠子参规范化栽培技术[J]. 陕西农业科学,2014,60(8):127.
[3] 王小平,白吉庆,王亚恒,等. 配伍对珠子参中4个皂苷类成分药代动力学的影响[J]. 中国实验方剂学杂志,2017,23(6):111.
[4] 刘爱华, 石孟琼, 杨文雁, 等. 珠子参总皂苷对大鼠心肌缺血/再灌注损伤的保护作用及机制研[J].中国临床药理学与治疗学, 2013, 18(11):1224.
[5] 宋小妹,李林,杨光明.不同产地珠子参中竹节皂苷IVa含量测定[J].中国中药杂志,2010,35(7):885.
[6] 宁张弛,刘振丽,宋志前. HPLC-QqQ-MS测定珠子参中5种皂苷类成分的含量[J]. 中国中药杂志,2016, 41(5):874.
[7] 时晓磊, 王加付, 姚华, 等. 珠子参化学成分分析[J]. 高等学校化学报,2013, 34(2):381.
[8] 张绍鹏, 金健, 胡炳雄, 等. 珍稀药用植物珠子参的转录组测序及分析[J]. 中国中药杂志, 2015, 40(11):2084.
[9] Han J Y, In J G, Kwon Y S, et al. Regulation ofginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng[J]. Phytochemistry, 2010, 71(1): 36.
[10] Han J Y, Kwon Y S, Yang D C, et al. Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng [J]. Plant Cell Physiol, 2006, 47(12): 1653.
[11] 罗志勇, 陆秋恒, 刘水平, 等. 人参植物皂苷生物合成相关新基因的筛选与鉴定[J]. 生物化学与生物物理学报, 2003, 35(6): 554.
[12] 吴亚运, 赵小龙, 陈平, 等. 珠子参β-香树素合成酶基因的克隆和生物信息学分析[J]. 生物技术通报, 2017, 33(2):109.
[13] 黄文静,谢培,杨洁, 等. 珠子参皂苷合成途径三个关键酶基因CAS、DS和β-AS时空表达分析[J]. 中国农学通报, 2017, 34(7):31.
[14] 赵新礼. 珠子参生态学和生物学特性的研究[J]. 现代中药研究与实践, 2015, 29(6):14.
[15] 赵仁, 赵毅, 何双凌, 等. 珠子参生产技术标准操作规程(SOP)[J].中国现代中药, 2014, 16(7): 556.
[16] Schmittgen T D, Livak K J. Analyzing real-time PCR databy the comparative CT method [J]. Nat Protoc, 2008, 3(6): 1101.
[17] 李凤英, 王英姿, 王晴, 等. 正交试验法优化珠子参总皂苷提取工艺[J]. 北京中医药大学学报, 2016,36(2):151.
[18] Haralampidis K, Trojanowska M, Osbourn A E. Biosynthesis of triterpenoid saponins in plants[J]. Adv Biochem Eng Biot,2002, 75:31.
[19] 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.
[20] 杨金玲, 高丽丽, 朱平. 人参皂苷生物合成研究进展[J]. 药学学报, 2013, 48(2):170.
[21] 明乾良,韩婷,黄芳, 等. 人参皂苷生物合成途径及其相关酶的研究进展[J]. 中草药, 2010, 41(11): 1913.
[22] 吴琼, 周应群, 孙超, 等. 人参皂苷生物合成和次生代谢工程[J]. 中国生物工程杂志, 2009,29(10):102.
[23] 黄文静, 孙晓春, 王楠, 等. 遮阴对珠子参生长发育及光合生理特性的影响[J]. 中国农学通报, 2017, 34(5):49.
[24] 黄文静, 王楠, 李铂, 等. 不同栽培年限珠子参在不同生长期的光合特性及保护酶活性研究[J]. 中国现代中药, 2017,19(10):1415.
[25] 徐克章, 张美善, 武志海, 等. 人参不同生育期叶片光合作用变化的研究[J]. 作物学报, 2006, 32(10): 1519.
[26] 张治安, 徐克章, 任跃英, 等. 光照条件对参株碳水化合物和人参皂甙含量的影响[J]. 吉林农业大学学报,1994, 16(3):15.