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

Real-time fluorescent quantitative PCR detection of key enzyme genes expression of alkaloid biosynthesis promoted by endophytic fungal elicitor in Sophora alopecuroides

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.