Comparision Of Defense Chemicals Of Rice Plants Infested By Different Herbivores And Setup Of Rice Lines With Antisense Inhibition Of Defense-related Gene OPR1 Or PLD3

Previous study showed that infestation by herbivores with different feeding habitats, the rice brown planthopper Nilaparvata lugens, the rice stembore Chilo suppressalia and the rice leaf folder Cnaphalacrosis medinais, activated different signal molecules in rice plants. Moreover, Chilo.suppressalia infestation up-regulated the expression levels of the rice genes OPR1 and PLD3. Here we analysed the changes in defense chemicals of rice plants infested by the three herbivores or induced by different signal molecules in order to illustrate the differences in defense chemicals of rice plants after attack by herbivores with different feeding habitats and their relation to the signal molecules that were differetly activated by the herbivores. We also set up rice lines with antisense inhibition of OPR1 or PLD3 gene by using Agrobacterium-mediated transformation system, which is the first step to elucidate the functions of the two genes in the defense responses of rice plants. The results are as follows:Totally, there were 13 chemicals in rice plants whose levels were increased after infestation by the three herbivores, which included momilactone A, momilactone B, and eleven unkown chemicals. The induced rice defense responses, however, were obviously different among the three herbivores. C. suppressalia infestation significantly increased the levels of the two diterpenoids, momilactone A and momilactone B, while N. lugens and C. medinais infestation did not. In other unknown eleven chemicals, N. lugens infestation enhanced the concentrations of five chemicals in stems, but had no effect on chemicals in leaves; C. suppressalia infestation resulted in increases in levels of seven chemicals in stems and five chemicals in leaves; infestation by C. medinais increased three chemicals in stems and five in leaves. During these chemicals, there was only one compound whose level could be increased after infestation by any one of the three herbivores; four compounds whose levels could be enhanced by N. lugens or C. suppressalia infestation; two chemicals by N. lugens or C. medinais infestation; three chemicals by C. suppressalia or C. medinais infestation. Using these 13 chemicals as indicators, we analysed the changes in defense chemicals of rice plants after treatment by one of the four signal molecules, jasmonic acid (JA), salicylic acid (SA), ethylene (ET) and hydrogen peroxide. The result showed that there were only 6 of the 13 chemicals whose levels were increased after treatment by the signals. JA, SA or ET treatment resulted in increases in concentrations of four chemicals in rice stems or leaves, and hydrogen peroxide treatment enhanced levels of five chemicals. None of the profiles of defense chemicals of rice plants infested by any one of the three herbivore was consistent with that induced by any signal molecule. This suggests that the defense responses of rice plants induced by N. lugens, C. suppressalia or C. medinais might be a result of cross-talk of multiple signal molecules.According to the published sequence, fragments of the rice gene OPRl and PLD3 were PCR amplified and cloned in pMD19-T vector respectively. After digestion with BamHI and KpnI, BamHI and SmalI respectively, the 730bp or 1000 bp resulting fragment was cloned in pCambia1301, yielding the transformation vector pCambia1301-OPR1, pCambia1301-PLD3 that contained the hygromycin resistance gene hpt as a selectable marker. On the T-DNA of the resulting binary plant transformation vector, the OPR1 or PLD3 gene fragment was present in an antisense orientation downstream from 35S promoter and upstream of terminators, thus enabling the transcription of OPR1 or PLD3 antisense RNA. By using Agrobacterium-mediated transformation system, rice lines with antisense inhibition of the gene OPR1 or PLD3 were set up, which lays the foundation for elucidating the function of the genes OPR1 and PLD3 in the defense responses of rice plants.