| The genomic DNA extracted by different methods from the fresh leaves of wheat seedlings(Jin mai NO.8), could be used as template to obtain an optimal system for RAPD analysis. On this basis, the damaged wheat seedlings induced by enhanced ultraviolet-B (10.08 kJ·m-2·d-1) radiation and the control ones were made for RAPD analysis to study the effect of UV on genomic DNA in wheat. Besides, three physiological indicators in wheat exposed to UV-B radiation were determined to study preliminary the mode of wheat seedlings to counteract UV radiation which promoted ROS generation. The results showed that:(1)The total DNA could be successfully extracted from the leaf tissue of wheat with advanced CTAB method. The extraction could be used for RAPD analysis.(2)DNA hyperchromicity in wheat seedlings strikingly declined with increasing UV radiation dose. This result indicated that UV-B stress could cause cross-linking between the two strands of DNA which was positively correlated with the radiation dose.(3) During the research, the main parameters in polymerase chain reaction such as template DNA,primers,dNTPs and Taq polymerase, were studied by setting gradient in order to establish an optimized RAPD reaction system. The results showed the optimum conditions in 25μL volume: 20ng template, 0.5μmol/L primers, 200μmol/L dNTPs, 0.75U Taq polymerase. The optimal amplification program was as follows: 2min at 94℃, followed by 30 cycles of 30s at 94℃, 30s at 38℃, 30s at 72℃, and then a final extension at 72℃for 10min.For further study, the PCR products could be stored in 4℃for a long time.(4) Under the optimum conditions,twelve primers were screened from 50 random primers, which could obtain clear, high polymorphism and reproducible RAPD products. The result showed that different bands were amplified by seven primers between the control group and UV-induced group. One specific band of 2,144bp occurred in UV-B treatments by S22, and brightness of the band at 1,470bp enhanced. Besides, other band patterns were basically consistent with the control ones. S33-1,619,S40-2,082,S123-1,071 and S160-813 also appeared in the UV-B treatments. While the fragments of S158-698 and AW78254-1,128 disappeared in UV-induced group and brightness of S158-1,128 increased in the control group. It came to a conclusion, a certain dosage of UV-B radiation could cause mutation of DNA molecules.(5)Although the effect of UV-B radiation on wheat was unstable, the two different fragments S22-2,144 and S40-2,082 which were amplified in this experiment appeared repeatly in different batches of wheat. The phenomenon illustrated that UV-induced DNA mutation also existed inevitability. It was also speculated that the specifical bands may be related with the molecular mechanism of DNA damage induced by UV-B.(6)Two above-mentioned specifical fragments, S22-2,144 and S40-2,082, were successfully recoveried with the improved method of TaKaRa Agarose Gel DNA Purification Kit Ver.2.0. The recoveried products were separated in 1.2% agarose gels and visualized by ethidium bromide staining. Two clear bands were gained but brightness of them was weaker, so it was very necessary to obtain an economic and effective recovery method to promote the concentration of DNA for further sequencing.(7)The determination results showed that the concentration of O2.- was measured in wheat cells before UV exposure. Compared to control samples, the content of O2.- strikingly rised with increasing radiation dose or treatment days, and the dose-effect was more pronounced. The level of flavonoids was positively correlated with the radiation dose and treatment days , which was similar to the variation trends of O2.-. A significant change in the content of polyphenols was observed in wheat before and after UV-radiation. Accompanied by the rise of UV dosage, the production of polyphenols in wheat gradually increased and then decreased after reaching a peak when exposed to UV-radiation of 13.5 kJ·m-2·d-1. With the increment of treatment days, the total polyphenol content rised overall, while the concentration of polyphenols in wheat induced by the maximal dose of UV-radiation (17.3 kJ·m-2·d-1) was lower than that in the group treated by the medium dose of UV-radiation (13.5 kJ·m-2·d-1) from the fourth to sixth treated day.To sum up ,above results led us to believe that UV radiation primarily induced secondary metabolites such as flavonoids and polyphenols accumulation rapidly to absorb UV directly in order to resist the damage caused by oxidation.
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