Functional Analysis Of The Gene Encoding The Key Enzyme PAL Assiociated With Weed-suppressive Ability In Allelopathic Rice Using RNAi

Allelopathic rice PI312777 was used as experiment material to prove functional properties of allelopathy in rice associated with function of PAL gene, and to reveal the process and mechanism of molecular ecology in rice allelopathy. The genome DNA was extracted from leaves of allelopathic rice PI312777. A fragment was produced by polymerase chain reaction using the specific primers designed on the conservative sequence of PAL gene in GenBank. The amplified fragment was cloned to pMD-18 T easy vector, and it was verified by sequencing. A DNA fragment with the length of 320bp of PAL gene was obtained. To construct RNAi expression vector, PTCK303-S-A, two copies of the 320bp fragment were further inserted into a expression vector, PTCK303, in an inverted—repeat orientation at both ends of intron. The vector was transferred into rice by Agrobacterium tumefaciens-mediated method. The gene fragment had been integrated into genomic DNA of PI312777 successfully by PCR detection and the GUS staining. The result of real-time fluorescence quantitative PCR showed the expression level of PAL gene in transformed PI312777 rice under normal nitrogen supply and low nitrogen supply was 70%and 75% of that of nontransformed PI312777 rice. The suppression rate of PAL gene is respectively 30% and 25%. PAL gene of transformed PI312777 rice under low nitrogen treatment was upregulated by 2. 12 fold in contrast to nontransformed PI312777 rice under normal nitrogen supplies. PAL gene of transformed PI312777 rice exposed to low nitrogen supply was upregulated by 2. 32 fold in contrast to transformed PI312777 rice under normal nitrogen supply. Analysis of Phenylalanine ammonia lyase activitity indicated the PAL enzyme activity of transformed PI312777 rice under normal nitrogen supply decreased 64% compared with nontransformed PI312777 rice at the same condition, suggesting that the gene had been interfered successively. The result obtained from the bioassay confirmed that allelopathic potential of the transformed rice and its counterparts under different nitrogen supplies was significant, performing that the dry weight of barnyardgrass cocultured with the transformed PI312777 rice line under low nitrogen treated solution was higher than that with the nontransformed PI312777 rice accession,and non-allelopathic rice accession, Lemont under low nitrogen treated solution. Inhibition rate of barnyardgrass mediated by the transformed PI312777 rice line under low nitrogen treated solution was much lower than that by the nontransformed PI312777 and non-allelopathic rice accession Lemont under low nitrogen treated solution. The same tendency was found in the case cocultured in normal nitrogen treated solution. This proved that functional properties of allelopathy in rice were associated with function of PAL gene.