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.
ABSTRACT
Objective: To investigate the signal molecules and signal transduction involved in endophytic fungal elicitor-induced atractylodin biosynthesis and the effect of an endophytic fungal elicitor on the key enzyme activity in Atractylodes lancea. Methods: Content changes of nitric oxide (NO), salicylic acid (SA), and atractylodin were detected under the endophytic fungal elicitor treatment by plant cell suspension culture technology. Results: The endophytic fungal elicitor remarkably promoted NO burst and the biosynthesis of SA and atractyodin by activating nitric oxide synthase (NOS), phenylalanine ammonia lyase (PAL), and acetyl coenzyme A carboxylase (ACC), respectively. NOS inhibitor PBITU could inhibit the NO and SA accumulation and the atractyodin biosynthesis induced by the elicitor. And atractyodin biosynthesis could also be triggered by exogenous NO or SA. The results indicated that NO and SA were the necessary signal molecules and NO burst was mediated by NOS induced by endophytic fungal elicitor. NO quencher cPITO could effectively remove NO burst in A. lancea cell induced by endophytic fungal elicitor and notably inhibit the biosynthesis promotion of SA and atractyodin in A. lancea cell induced by endophytic fungal elicitor. Exogenous SNP could reverse the cPITO inhibition on the activity of PAL and ACC and the synthesis of SA and atractylodin. This suggested that NO was an upstream signal molecule mediated endophytic fungal elicitor to accelerate the biosynthesis of SA and atractyodin. Conclusion: Endophytic fungal elicitor mediated through NO followed by SA could promote atractyodin biosynthesis by activating ACC in A. lancea.
ABSTRACT
Objective To establish the suspension cell line of Atractylodes lancea and to study the effect of two endophytic fungal elicitors on its essential oil production.Methods The essential oil was extracted by using ultrasonic wave after suspension cell was treated with endophytic fungal elicitors.Then,the determination of four compounds(atractylone,hinesol,?-eudesmol,and atractylodin) was carried out by gas chromatography.Results By testing in various conditions,the suspension cell line with a rapid growth rate was established.Its highest biomass(6.95 g/L) was obtained on day 21.?-Eudesmol was the only detection in the control suspension cell,and its highest content(17.469 ?g/g) was also reached on day 21.The effect of crude elicitors of two endophytic fungi(belong to Cunninghamella sp.and Gilmaniella sp.respectively,named AL4 and AL12) on the cell growth and the production of essentia1 oil were investigated.Overall AL4 elicitor got better effect.When suspension cell of 14-day-old cultures was exposed to AL4 elicitor(carbohydrate 20 mg/L medium) for 7 d,the biomass increased 3.31% over the control,and the four compounds(atractylone: 14.715 ?g/g,hinesol: 28.395 ?g/g,?-eudesmol: 38.794 ?g/g,and atractylodin: 8.310 ?g/g) were all detected.Among them,the content of ?-eudesmol reached 2.22 times as much as the control.Conclusion The cell growth and the accumulation of essential oil of A.lancea could also be promoted by adding crude elicitors of the endophytic fungi AL4 and AL12.