Identification And Cloning Of Nilaparavata Lugens Inducible Rice Promoters

Rice is one of the most important food crops in the world, especially for Asian people. The pest control has always been a focus in rice breeding. With the development of transgenic technology, the promoter, as an important component of expression vector construction in genetic engineering, has also been treated seriously. It may control the position, time and intensity of gene expression. Currently, some brown planthopper (BPH) resistance genes have been identified or isolated from the natural rice germplasms. The rice BPH inducible promoter can be used to drive the expression of the BPH resistance genes. The fused resistance gene with a BPH inducible promoter will be up-regulated when the transgenic rice is infested by BPH. It will enhance the resistance rice to BPH and meanwhile avoid the excessive consumption of material and energy in transgenic rice with better quality and biosafety. The objective of this study is to isolate the BPH inducible promoter from rice genome, providing an effective tool for the transgenic breeding of insect-resistance. This study obtained the following results:1. Based on the expressional profile data of rice whole genome under BPH infestation condition by Li Changyan and the expressional profile data of rice whole genome of all organs and tissues during the whole rice growth by our laboratory, eight BPH inducible and green tissue specific rice genes are chosen. The gene expressional patterns of these eight genes were validated via RT-PCR analysis. The promoter regions of these eight genes were isolated from either TN1 a BPH susceptive variety or RH a BPH resistant variety. These promoters are referred to as PTG1, PTG2, PTG3, PTG4, PTG5, PTG1, PRG4 and PRG6, respectively.2. The eight promoters were ligated with the GUS reporter gene in the binary Ti vector DX2181b respectively to form the final expression vectors. The final expression vectors were transformed into to the japonica rice variety Zhonghua 11. By far, the transgenic plants with six promoters PTG1, PTG2, PTG3, PTG4, PTG1 and PRG4 have been acquired. While the transformation of the expression vectors containing the promoters PTG5 and PRG6 are still ongoing. The expressional pattern of six promoters PTG1, PTG2, PTG3, PTG4, PRG1 and PRG4 were determined by using transgenic rice plants under BPH feeding or Jasmonic acid (JA) treatment. According to the qRT-PCR analysis of the expression of the GUS report gene, three rice promoters PTG3, PRG1 and PRG4 showed application potential.3. The expressional level of GUS report gene driven by the promoter PTG3 increase by up to 4.38 times and 3.17 times under BPH feeding and JA treatment respectively. Moreover, PTG3 is a strong promoter because the histochemical staining of PTG3 transgenic rice showed intense GUS activity.4. According to the gene chip data, the expressional level of the endogenous gene of PRG1 increases by 97 times in the BPH resistant variety RH after BPH infestation. The qRT-PCR analysis showed that the endogenous gene of PRG1 increases by 968 times in RH under BPH feeding. By detecting the transcript level of PRG1+GUS fusion gene, GUS transcript level increases by up to 140.50 times under BPH infestation, but only increase slightly under treatment. However, although induced strongly by BPH.infestation, the PRG1 has a weak whole promoter activity because the histochemical staining could not detect the GUS activity even under the induction of BPH feeding.5. Although PRG4 is induced by both BPH feeding and JA treatment, it seems to be preferentially induced by BPH feeding. The GUS transcript of PRG4+GUS fusion gene increases by up to 22.08 times under BPH feeding, but increases by up to 4.46 times under JA treatment. The histochemical staining analysis showed that PRG4 is also a weak promoter.In conclusion, the BPH inducible promoter can be induced by specific condition and therefore has great application potential for the crop breeding insect-resistance and disease-resistance with the reduction of chemical pollution and the increase of crop yield. Moreover, it provides good research resources for relevant promoters and genes study.