苦豆子内生真菌诱导子促进宿主生物碱合成关键酶基因表达的荧光定量PCR检测
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摘要
目的建立荧光定量PCR(qRT-PCR)检测苦豆子内生真菌诱导子促进宿主生物碱合成关键酶—赖氨酸脱羧酶(LDC)基因的表达的方法。方法根据苦豆子LDC和Lectin基因序列设计目的基因引物QLDC-F/QLDC-R和内参基因引物Lectin-F/Lectin-R;以5倍梯度稀释的c DNA作为标准样品,建立目的基因QLDC-F/QLDC-R和内参基因Lectin-F/Lectin-R的标准曲线,优化qRT-PCR反应体系与反应条件,分析比较半定量PCR与qRT-PCR的灵敏度;在苦豆子内生真菌NDZKDF13诱导子不同诱导时间下,高效液相色谱(HPLC)测定宿主氧化苦参碱(oxymatrine,OMA)的含量,qRT-PCR检测LDC基因的表达量,分析在内生真菌诱导下LDC基因与OMA合成积累的关系。结果在qRT-PCR体系中c DNA质量浓度为200 ng/μL,退火温度为61℃时检测结果最好;构建的目的基因和内参基因标准曲线,其循环阈值与模板浓度均呈良好的线性关系,扩增效率都在99%以上,灵敏度是半定量PCR的25倍;在内生真菌NDZKDF13诱导子诱导作用下,宿主LDC基因的表达量在第6天达到峰值,为对照的25.58倍;OMA含量的增加滞后于LDC基因表达量的变化,在诱导子处理第9天达到最高峰。结论成功将qRT-PCR技术应用于苦豆子的功能基因研究。通过对各种条件的优化探索,建立了准确和简单易行的检测苦豆子的功能基因表达的平台。
Objective To establish a method of detecting the expression of Lysine decarboxylase(LDC) —a key enzyme for the synthesis of alkaloid in the host promoted by the endophytic fungal elicitor of Sophora alopecuroides by using real-time fluorescence quantitative PCR(qRT-PCR). Methods Target gene primers QLDC-F/QLDC-R and reference gene primers Lectin-F/Lectin-R were designed according to LDC and Lectin gene sequences of S. alopecuroids; Five-fold gradient dilution of c DNA was used as the standard sample for the construction of the standard curve of target gene and the reference gene. Reaction system and reaction conditions of qRT-PCR were optimized, and the sensitivity of semi-quantitative PCR and qRT-PCR were analyzed and compared. Under different eliciting time of endophytic fungal elicitors NDZKDF13 of S. alopecuroides, the content of oxymatrine in the host was determined by HPLC, the expression of LDC gene was detected by qRT-PCR, and the relationship between LDC gene expression and the accumulation of OMA was analyzed. Results The results of qRT-PCR were better when the c DNA content in the system was 200 ng/μL and the annealing temperature was 61 ℃. The standard curve of the target gene and the reference gene was constructed, in which the cycle threshold and template concentration showed a good linear relationship, the amplification efficiency was above 99%, and the sensitivity was 25 times that of semi-quantitative PCR. Under the induction effect of endophytic fungal elicitor NDZKDF13, expression of host LDC gene reached the peak on the 6 th day, which was 25.58 times that of the control. The increase of OMA content lagged the change of the LDC gene expression and reached the highest amount on the 9 th day after the induction. Conclusion The qRT-PCR technique was successfully applied to the functional gene research of S. alopecuroides. Through the optimization of various conditions, a platform for accurate and simple detection of functional gene expression in S. alopecuroides was established.
Objective To establish a method of detecting the expression of Lysine decarboxylase(LDC) —a key enzyme for the synthesis of alkaloid in the host promoted by the endophytic fungal elicitor of Sophora alopecuroides by using real-time fluorescence quantitative PCR(qRT-PCR). Methods Target gene primers QLDC-F/QLDC-R and reference gene primers Lectin-F/Lectin-R were designed according to LDC and Lectin gene sequences of S. alopecuroids; Five-fold gradient dilution of c DNA was used as the standard sample for the construction of the standard curve of target gene and the reference gene. Reaction system and reaction conditions of qRT-PCR were optimized, and the sensitivity of semi-quantitative PCR and qRT-PCR were analyzed and compared. Under different eliciting time of endophytic fungal elicitors NDZKDF13 of S. alopecuroides, the content of oxymatrine in the host was determined by HPLC, the expression of LDC gene was detected by qRT-PCR, and the relationship between LDC gene expression and the accumulation of OMA was analyzed. Results The results of qRT-PCR were better when the c DNA content in the system was 200 ng/μL and the annealing temperature was 61 ℃. The standard curve of the target gene and the reference gene was constructed, in which the cycle threshold and template concentration showed a good linear relationship, the amplification efficiency was above 99%, and the sensitivity was 25 times that of semi-quantitative PCR. Under the induction effect of endophytic fungal elicitor NDZKDF13, expression of host LDC gene reached the peak on the 6 th day, which was 25.58 times that of the control. The increase of OMA content lagged the change of the LDC gene expression and reached the highest amount on the 9 th day after the induction. Conclusion The qRT-PCR technique was successfully applied to the functional gene research of S. alopecuroides. Through the optimization of various conditions, a platform for accurate and simple detection of functional gene expression in S. alopecuroides was established.
引文
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[9]崔颖,王秀娟,高山,等.实时荧光定量PCR技术在植物中的应用[J].湖北农业科学,2015,54(13):3073-3077.
[10]向蓓蓓,李晓雪,王勇,等.川西獐牙菜牻牛儿基焦磷酸合成酶基因的克隆及表达分析[J].中草药,2017,48(5):962-970.
[11]李清,李标,郭顺星.金钗石斛焦磷酸甲羟戊酸脱羧酶基因的克隆及表达分析[J].基因组学与应用生物学,2018,37(2):2223-2229.
[12]高媛,孙牧笛,徐全智,等.苦豆子内生真菌对宿主培养物生长及喹诺里西啶类生物碱合成的影响[J].干旱地区农业研究,2017,35(3):212-218.
[13]周星辰.苦豆子内生真菌遗传多样性及其产喹诺里西啶生物碱菌株的筛选[D].银川:宁夏大学,2014.
[14]杨毅.苦豆子赖氨酸脱羧酶基因克隆及表达分析[D].银川:宁夏大学,2015.
[15]张海波.适合转基因大豆GTS 40-3-2复合PCR检测标准分子研制及番茄内标准基因国际协同验证[D].上海:上海交通大学,2008.
[16]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T))method[J].Methods,2001,25(4):402-408.
[17]唐永凯,贾永义.荧光定量PCR数据处理方法的探讨[J].生物技术,2008,18(3):89-91.
[18]于春梅,刁有祥,唐熠,等.坦布苏病毒荧光定量RT-PCR方法的建立[J].中国农业科学,2012,45(21):4492-4500.
[19]Gutierrez L,Mauriat M,Pelloux J,et al.Towards a systematic validation of references in real-time RT-PCR[J].Plant Cell,2008,20(7):1734-1735.
[20]周晓慧,刘军,庄勇.喀西茄内参基因实时荧光定量PCR表达稳定性评价[J].园艺学报,2014,41(8):1731-1738.
[21]曾松荣,徐成东,王海坤,等.药用植物内生真菌及其具宿主相同活性成分的机制初探[J].中草药,2000,31(4):68-70.
[22]徐茂军,董菊芳,朱睦元.NO参与真菌诱导子对红豆杉悬浮细胞中PAL活化和紫杉醇生物合成的促进作用[J].科学通报,2004,49(7):667-672.
[23]徐茂军,董菊芳,朱睦元.NO通过水杨酸(SA)或者茉莉酸(JA)信号途径介导真菌诱导子对粉葛悬浮细胞中葛根素生物合成的促进作用[J].中国科学C辑:生命科学,2006,36(1):66-75.
[24]杨致荣,陈钊,李润植.茉莉素介导的长春花生物碱次生代谢转录调控机制[J].中国生物化学与分子生物学报,2014,30(6):533-542.
[25]高媛.苦豆子悬浮细胞培养及其内生真菌对宿主喹诺里西啶生物碱合成积累的影响[D].银川:宁夏大学,2016.
[2]陶金华,汪冬庚,濮雪莲,等.H2O2介导内生真菌诱导子促进茅苍术细胞HMGR的活化和β-桉叶醇的生物合成[J].中草药,2013,44(19):2740-2744.
[3]石岳香,周敏,杨华,等.内生真菌和诱导子对长春花悬浮细胞及生物碱合成的影响[J].现代生物医学进展,2009,9(5):886-889.
[4]胡会平,刘建慧,孙鑫,等.苦参碱及衍生物的生物活性和合成研究进展[J].鲁东大学学报:自然科学版,2015,31(4):339-345.
[5]Hartmann T,Schoofs G,Wink M.A chloroplast-localized lysine decarboxylase of Lupinus polyphyllus:The first enzyme in the biosynthetic pathway of quinolizidine alkaloids[J].Febs Lett,1980,115(1):35-38.
[6]Schoofs G,Teichmann S,Hartmann T,et al.Lysine decarboxylase in plants and its integration in quinolizidine alkaloid biosynthesis[J].Phytochemistry,1983,22(1):65-69.
[7]Bunsupa S,Katayama K,Ikeura E,et al.Lysine decarboxylase catalyzes the first step of quinolizidine alkaloid biosynthesis and coevolved with alkaloid production in leguminosae[J].Plant Cell,2012,24(3):1202-1216.
[8]杨毅,田蕾,刘萍,等.苦豆子赖氨酸脱羧酶基因表达与苦参碱和氧化苦参碱含量的关系[J].中国药学杂志,2015,50(10):846-849.
[9]崔颖,王秀娟,高山,等.实时荧光定量PCR技术在植物中的应用[J].湖北农业科学,2015,54(13):3073-3077.
[10]向蓓蓓,李晓雪,王勇,等.川西獐牙菜牻牛儿基焦磷酸合成酶基因的克隆及表达分析[J].中草药,2017,48(5):962-970.
[11]李清,李标,郭顺星.金钗石斛焦磷酸甲羟戊酸脱羧酶基因的克隆及表达分析[J].基因组学与应用生物学,2018,37(2):2223-2229.
[12]高媛,孙牧笛,徐全智,等.苦豆子内生真菌对宿主培养物生长及喹诺里西啶类生物碱合成的影响[J].干旱地区农业研究,2017,35(3):212-218.
[13]周星辰.苦豆子内生真菌遗传多样性及其产喹诺里西啶生物碱菌株的筛选[D].银川:宁夏大学,2014.
[14]杨毅.苦豆子赖氨酸脱羧酶基因克隆及表达分析[D].银川:宁夏大学,2015.
[15]张海波.适合转基因大豆GTS 40-3-2复合PCR检测标准分子研制及番茄内标准基因国际协同验证[D].上海:上海交通大学,2008.
[16]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T))method[J].Methods,2001,25(4):402-408.
[17]唐永凯,贾永义.荧光定量PCR数据处理方法的探讨[J].生物技术,2008,18(3):89-91.
[18]于春梅,刁有祥,唐熠,等.坦布苏病毒荧光定量RT-PCR方法的建立[J].中国农业科学,2012,45(21):4492-4500.
[19]Gutierrez L,Mauriat M,Pelloux J,et al.Towards a systematic validation of references in real-time RT-PCR[J].Plant Cell,2008,20(7):1734-1735.
[20]周晓慧,刘军,庄勇.喀西茄内参基因实时荧光定量PCR表达稳定性评价[J].园艺学报,2014,41(8):1731-1738.
[21]曾松荣,徐成东,王海坤,等.药用植物内生真菌及其具宿主相同活性成分的机制初探[J].中草药,2000,31(4):68-70.
[22]徐茂军,董菊芳,朱睦元.NO参与真菌诱导子对红豆杉悬浮细胞中PAL活化和紫杉醇生物合成的促进作用[J].科学通报,2004,49(7):667-672.
[23]徐茂军,董菊芳,朱睦元.NO通过水杨酸(SA)或者茉莉酸(JA)信号途径介导真菌诱导子对粉葛悬浮细胞中葛根素生物合成的促进作用[J].中国科学C辑:生命科学,2006,36(1):66-75.
[24]杨致荣,陈钊,李润植.茉莉素介导的长春花生物碱次生代谢转录调控机制[J].中国生物化学与分子生物学报,2014,30(6):533-542.
[25]高媛.苦豆子悬浮细胞培养及其内生真菌对宿主喹诺里西啶生物碱合成积累的影响[D].银川:宁夏大学,2016.