ABSTRACT
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 cDNA 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 cDNA 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 6th 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 9th 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.