| Polygonum Cuspidatum is a kind of herbage belongs to polygonaceae. It is a Chinese traditional medicine, Mainly in the East, South, Gansu,Sichuan, Yunnan and other places. The extracts of Polygonum have anti-inflammatory and prevention of cardiovascular disease and other pharmacological activities, including the main active ingredient is resveratrol and emodin. As a phytoalexin,it not only can improve the plant to UV, the resilience of fungi and other injuries,but also can Protective effect on the cardiovascular system. This study dealt with the young stalks of perennial Polygonum Cuspidatum as explants and treating the callus induction as materials.By ultraviolet light and put into the darkness for processing and additional H2O2, study by the callus growth and changes in resveratrol content. By semi-quantitative RT-PCR amplification discussion the gene expression of the increased levels of RS. The studying results showed as follows1. The changes of H2O2 content by different ultraviolet radiation and different sampling time to Polygonum cuspidatum callusThe overall trend of the content of H2O2 by short time sample group is first increased and then decreased,have some fluctuation, the long time sampling callus group's H2O2 content for the overall trends of modest fluctuations.The peaks of short sample group is 2hours and 4hours.The H2O2 content of all treatments are higher than ck, and the 4th treat is higher levels of H2O2. At 2hour, the peak for 4 was 2.68nmol·g-1, increased 54% by CK. At 4 hour, the peak for 4 was 2.84nmol·g-1, increased 37% by CK. The sampling time had little effect on H2O2 content for the longtime sampling group. To the latter, the trend is a certain level of volatility.2. The forms of the callus and the content of the callus's resveratrol by different times UV and different sampling time.After the ultraviolet radiation the callus of polygonum at different times, no significant change in callus in 5 hours, the color is still green, shape and fluffy. Four days on, the callus are aging, slow growth, aging occurs earlier than CK about 8 days.The Res content in each treatment is erratic ups and downs in short time(1-5h).4days later,the Res levels increased significantly, and reached a peak at 12day. the Res content of 3 treatment was significantly different of CK.After the 12day, the Res content of all treatments decreased,The whole tread of Res content,The Res content of third treatment has been at a high level.3. The changes of the forms and Res content by the callus which plus H2O2 and H2O2 quencherJoin the H2O2 quencher and the H2O2 at the content which is the highest in the callus to the medium. After inoculation,, no significant change by callus in 5 hours,the color is still green. From 4 days on, the callus which added H2O2 aging,and the form of the callus which medium added H2O2 quencher like CK, no significant change by callus in 5 hours,didn't aging at 4 day,from 12 days on,it aging.The sampling time of the callus which added H2O2 and H2O2 quencher in medium is still shorttime (1-5h) and longtime (4-20d).Determine the Res of callus by HPLC, The results showed that the content of Res by external H2O2 group in 1-5h and 4d are little higher than ck, from 4 days on,the content of Res are lower than CK, And in the 12d was extremely significant.The Res of callus which added H2O2 quencher were not significantly different with CK in 5hours,4-20days it Increased slightly, but significantly lower than CK4.Detect the gene expression levels of RS.Extracted RNA by the callus in each treat and testing the Integrity by the electrophoresis. The results showed that the 28S,18S,5.8S of three samples shows clearly,it means that the total RNA is good and complete, It meet the experimental requirements of the following RT-PCR amplification. The RNA is using for semiquantitative RT-PCR amplification, taking 1.2μl product of after reaction, add the Rnase scavenger to TAE as the buffer, electrophoresis 20mins by 90V/cm, Photographed and observation under ultraviolet light. All the three samples were amplified actin which is 600bp internal reference gene,and there is no difference in brightness and width. It shows that the expression of the reference gene is stability., there is a about 1200bp's specific fragment A above the 1000bp which from the highest Res content of group by using specific primers. But the control group and the group which join in the H2O2 is not amplified the fragment. Shows that, after UV irradiation, the level of the gene expression of RS has improved... |
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