Inheritance, Expression And Insects Resistance Of Protease Inhibitor Ⅱ Gene (PinⅡ) Driven By Different Promoters In Transgenic Rice

Proteinase inhibitors are proteins widely distributed in plants. In addition to their roles as storage protein, they are important defensive proteins against insects and microorganism. As a member of the serine proteinase inhibitor family, potato proteinase inhibitor II ( Pin II) resists against insects broadly and stably because it contains two reactive sites, one of which inhibits trypsin and the other of which inhibits chymotrypsin. In this study, Pin II gene was introduced into Japonica rice variety Nipponbare via Agrobacterium mediated transformation. Furthermore, inheritance, expression and insects resistance of Pin II driven by different promoters in transgenic rice were also investigated. Main results are as follows:1. Binary vectors pNAR301 ( ActI5'+PinII-2x+PinII3' ) , pNAR302 (UbiI5'+PinII-2x+NOS3') and pNAR303 (PinII5'+PinII-4x+PinII3') constructed in our lab were introduced into Japonica rice variety Nipponbare via Agrobacterium mediated transformation. Twelve regenerated shoots were obtained. Eight of them were positive by method of leaf assaying hygromycin resistance in vitro and PCR amplification. The ratio of positive plants was 67.7%.2. The inheritance of rice lines transformed by PinII under control of different promoters was investigated by analysis of hygromycin resistance, PCR and Southern blot. For segregation patterns of hygromycin phosphotransferase gene (hpt), 68.4% of the transgenic rice plants were conformed to a Mendelian ratio. Presence of the transgenes was determined by PCR analysis. Southern blot indicated that there was a high frequency of single-copy transgene integration in majority plants without rearrangement and the rate of single insertion was 63.6%. Consequently, it proved that Pin II had stably integrated into rice genome.3. The expression of Pin II in transgenic plants was investigated in the level of RNA and protein by northern blots and enzymatic assay. Northern blots confirmed that the transcripts of the foreign gene in transgenic rice plants were different and not related with the copy number of integrated transgene. But the inhibitory activity of Pin II against trysin was connected with gene promoters. Quantitative analysis of Pin II protein expressed in transgenic rice plants showed that Pin II protein in fresh leaves was 160 μg/g for ActI- Pin II-2x, 176μg/g for Ubi- Pin II-2x, and 104μg/g...