| Amaryllidaceae-type alkaloids is the important natural production of Amaryllidaceae,possessing pharmaceutically active.In order to analyze the TYDC gene of Lycoris Radiate regulation on the alkaloids accumulation in Lycoris Radiate shoots.In this study,the expression levels of TYDC gene,the enzyme activity of TYDC and the accumulation of alkaloids of Lycoris Radiate shoots under different growth conditions were investigated.Main results were concluded as below.1.The well reference gene selection.The reproducibility and reliable results of qRT-PCR is base on the use of suitable housekeeping or reference genes as internal standards.The expression levels of ACT,18 s rRNA,GADPH,P450 of Lycoris Radiate shoots under different growth condition were studied by qRT-PCR technology.The date was analyzed by GeNorm software.The result showed that the average expression stability value of reference gene M of the four genes from high to low is that P450 > GAPDH > 18 S rRNA > ACT.So the suitable reference gene for qRT-PCR was ACT gene.2.The relative expression levels of TYDC gene of Lycoris Radiate shoots under different growth condition were detection by qRT-PCR technology.The results indicated that the transcriptional level of TYDC in the treatment of tyrosine was increased.The expression level reached the pick at 48 h.The transcriptional level was ascended insignificantly after treated with low concentration MeJA.The highest expression level appeared at 24 h with treatment of 20 ?mol/L MeJA.The expression level was declined with the concentration of MeJA increasing.The transcriptional level of TYDC gene was inhabited with treating of 100 ?mol/L and 150?mol/L MeJA.3.The method for detection activity of TYDC was established.The reaction system of TYDC was include 0.1mol/L PBS,2 mmol/L tyrosine,0.2 mmol/L pyridoxal phosphate,0.5 mL crude enzyme,the total volume was 3 mL and the reaction time was 30 min.The activity of TYDC was showed by the content of tyramine.The concent of tyramin was detection by HPLC.Tyramine were separated on the Eclipse XDB-C18 reversed phase column(150 mm × 4.6 mm,5 ?m)by using 50 mmol/L of ammonium acetate aqueous solution(pH 4.5,adjusted with acetic acid)(A)and methanol(B)as mobile phase.The gradient was: 0-4 min A 96%,B 4%;4-20 min A 96-90%,B 4-10%.The flow rate was 1.0mL/min and the detection wavelength was set at 276 nm.The volume of injection was 10?L.4.The effect of precursor and elicitor on the activity of TYDC.Treatment with tyrosine,the activity of TYDC was increased.The activity of TYDC was the highest with addition of 150?mol/L tyrosine,and the activity declined with the concentration of tyrosine increasing.The activity of TYDC was observably increased after treating with low concentration of MeJA.And the activity markedly declined after addition of high concentration of MeJA.5.The content of alkaloids in Lycoris Radiate shoots was detected by HPLC.The results showed that tyrosine had effects on the accumulation of lycorine,lycoramine and galanthamine in shoot cultures.However,high concentrations of tyramine significantly increased the contents of the three alkaloids in the shoot cultures.Treatment with 150?mol/L of tyrosine,the levels of lycorine in the shoots was 349.86 ?g/g,it is 2.66 times more than the control culture.And addition of 200?mol/L of tyrosine,the accumulation of lycoramine and galanthamine were 391.53 ?g/g and 233.48 ?g/g,and these were 2.58-fold and2.80-fold higher than the control.Low concentrations of MeJA significantly increased the accumulation of the three alkaloids in the shoot cultures,whereas high concentrations of MeJA negatively affected the contents of lycorine,lycoramine and galanthamine in thecultures.The highest levels of the lycorramine were obtained upon treatment with 20?mol/L of MeJA,and were 2.74-fold,greater than the control cultures.And after treatment with 50?mol/L of MeJA,the accumulation of lycorine and galanthamine were 1.90-fold and 2.20-fold more than the control groups,respectively. |
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