Study On Protein Expression Variation Of Paeonia Suffruticosa In Vitro In The Period Of Rooting Inducement

The axillary buds of P.suffruticosa'Wulongpengsheng'were cultured as explants. And the basal part of stem of the plantlets was used as experimental material to established an suitable and efficient 2-DE procedure for proteome of basal part of stem of tree peony in vitro. In this research we tried to find the proteins related with rooting, so that we could study the rooting mechanism on the level of protein and molecular, and solve the rooting problem of tree peony in vitro, the situation of protein express variation of tree peony in vitro in the period of rooting elicitation was studied. The results are as follows:1. TCA/acetone precipitation was the best method of protein extraction for proteome of basal part of stem of tree peony in vitro. Different protein extraction methods were compared for the analysis of 2-DE system. The results showed that, sample prepared by ammonium acetate/methanol/phenol extraction only got 45 protien spots on the 2-DE map, and the spots were indistinct, having obvious streak appearance and low resolution. Method of ethanol/ether/acetone precipitation got 101 protien spots, but they were also not clear and had an obvious interference of crosswise and upright streaks. More stable repetitiveness, high clarity and regular shape protein spots(434) were obtained on the 2-DE map in the method of TCA/acetone precipitation. What was more this method was a little simpler and had good reproducibility.2. The suitable loading quantity for proteome of basal part of stem of peony in vitro was 1200μg. Extracted by the method of TCA/acetone, with IPG gel of pH 3-10(24cm)and 12%PAGE resolving gel, different loading quantities of protein sample (800μg,1000μg,1200μg) were used for 2-DE. When the loading quantity was 800μg, there were 188 protien spots on the 2-DE map, they were indistinct and had low resolution. The loading quantity of 1000μg had 273 protien spots,and they had the same problem of indistinction and low resolution. When the loading quantity was 1200μg, the proteins were separated well on the 12% SDS-PAGE gel, 562 protien spots were distinguished on the 2-DE map.3. The result of 2-DE maps for tree peony in different rooting period showed that: most of the proteins in the basal part of tree peony in vitro were in acidity. The main range of pI was 4 to 7.Only few proteins were in the alkaline area. The 2-DE maps for different period had a good similarity,but they still revealed some differences on the quantity and especially expression of protein spots. There were 3 protein spots up-regulated, 4 protein spots were down-regulated and 1 new protein spot appeared on the 2-DE map for the 5th day.4. Eight significant expression protein spots were identified by LC/ESI/ MS/MS,checked the information of peptide sequences in database.They were the kind of protein in chloroplast,ATP synthase beta subunit.ATP synthase was the crucial enzyme in the period of photosynthesis and energy metabolish for plant.βsubunits were the key part of ATP synthase, they were the catalytic sites for ATP synthesis. Inferred from the result, the expression variation of ATP synthase beta subunit was involved with the complicated energy metabolish in the period of taking adventitious root.