Cloning And Identification Of Nilaparavata Lugens Inducible Promoters And Bidirectional Promoters In Rice

Rice is the important food crops in china. As its yield and quality are greatly influenced by plant diseases and insect pests, it is critical to improve the resistance of diseases and insect pests. With the development of transgenic technology, the promoter, as an important part of expression vector, has become a hot spot in genetic engineering. The BPH inducible promoter can make the resistant gene specifically expressed in rice with BPH feeding, not only avoid the energy consumption caused by the constitutive promoter expression, but also enhance the rice resistance of BPH. The bidirectional promoter can drive two genes expressed in rice in the same time. This not only can avoid the gene silence caused by the using of homologous promoters, but also can make the rice have different resistance. In this study, four BPH inducible promoters have been cloned and the functions have been tested. The sequence between the four bidirectional gene pairs has been cloned and the functions have been tested. The main results were as follow:1. Based on the expressional profile data of rice whole genome under BPH infestation condition by Li Changyan, four BPH inducible genes were chosen after the test via RT-PCR analysis. These promoters were acquired from the genomic DNA of RH by PCR and named as PYM1, PYM2, PYM4 and PYM5, respectively.2. The four promoters were inserted into the promoter function analysis vector p DX2181 and transformed into the japonica rice variety Zhonghua 11. The transgenic plants with four promoters PYM1, PYM2, PYM4 and PYM5 have been acquired. The expressional pattern of four promoters PYM1, PYM2, PYM4 and PYM5 were determined by using transgenic rice plants under BPH feeding or acupuncture treatment. According to the q RT-PCR analysis, the expressions of the GUS report gene are raised after acupuncture treatment and BPH feeding. So the four promoters can be acupuncture treatment and BPH feeding induced expression.3. The expression efficiency of PYM1 is high and has no difference after acupuncture treatment and BPH feeding. The expressional level of GUS report gene increase by 2.04 times and 2.35 times under acupuncture treatment and BPH feeding. The expression efficiencies of PYM2 and PYM5 are low and have significant change after BPH feeding. The expressional level of GUS report gene drived by the promoter PYM2 increases by 1.84 times and 2.09 times under acupuncture treatment and BPH feeding. The expressional level of GUS report gene drived by the promoter PYM5 increases by 1.36 times and 6.07 times under acupuncture treatment and BPH feeding. The expression efficiency of PYM4 is very low and has no difference after acupuncture treatment and BPH feeding. The expressional level of GUS report gene increases by 2.37 times and 1.50 times under acupuncture treatment and BPH feeding.4. Four bidirectional gene pairs have been found in rice by bioinformatics technology. Their promoters were acquired from the genomic DNA of Minghui 63 by PCR and named as BDP1, BDP3, BDP4 and BDP5, respectively. Then inserted into the promoter function analysis vector p DX2181 in forward and reverse directions and transformed into the japonica rice variety Zhonghua 11. BDP1 was identified as an endosperm-specific promoter in transgenic rice plant by histochemical staining. BDP3, BDP4 and BDP5 can also drive the GUS report gene expressed in bidirections.5. The promoter fragment of BDP1 downstream extension of 123 bp from the end nearer the transcription start site. The new promoter named as BDP2 is inserted into the promoter function analysis vector p DX2181 in forward and reverse directions(named BDP21 and BDP22, respectively). The expression pattern of the BDP2 is the same as BDP1. And the expression efficiency of BDP21 and BDP22 are both higher than BDP1.