Study On Genetic Transformation Of Rice With Phenylalanine Ammonia-lyase

Phenylalanine Ammonia-lyase(PAL)is the key enzymes and the rate-limiting enzyme of the Phenylpropanoid metabolism, PAL is also the rate-limiting enzyme in the biosynthetic pathway of flavonoid, the activity of PAL is positively correlated with the formation of flavonoids. Flavonoids have anti-tumor, anti-cancer, anti-cardiovascular disease, antioxidant and anti-aging, immune regulation, anti- sugar leukemia and other important pharmacological effects.Because we can not synthetic flavonoid, we mainly uptake flavonoid from food.This study used the genetically engineering techniques to clone PAL gene from high-flavonoid rice"M-1". We had imported the PAL gene to other low-flavonoid rice by the method of Agrobacterium-mediated.The specificity and highly expression of PAL gene in the rice endosperm should rise the content of flavonoid, which can be used for breeding high-yield and high nutritional quality of rice lines. The main results include:1. The plant expression vector pCDMAR-PAL-Hyg was constructed, which contained two spereate T-DNA regions, One T-DNA region carried the selectable marker gene (Hyg), the other T-DNA region carried the PAL gene. The PAL gene was driven by Gt1 promoter which specificity express in rice endosperm, two sides of the PAL gene cassette with two direct repeat Rb7MARs of tobacco, in order to enhance the expression of PAL gene.2. The plant expression vector pCDMAR-PAL-Hyg was transformed to the embryonic tissue of"TG-9","MH86"and"527"by the method of Agrobacterium-mediated. We obtained 153"TG-9"transgenic rice, 27 of which were positive by PCR testing. We obtained 7"527"transgenic rice, 3 of which were positive by PCR testing.We obtained 3"MH86"transgenic rice, 1 of which was positive by PCR testing.We did single enzyme digested genomic DNA southern blot, identified that PAL gene has been transformed into rice. By PCR testing on"TG-9"T₁ generation transgenic rice, we had obtained some transgenic rice which only have PAL gene but no Hyg gene.3. We measured the content of flavonoids in"TG-9"positive transgenic rice on T₀ generation by the method of UV-Visible Spectrophotometer. Compared with CK, the content of flavonoids increased in 26 positive transgenic rice: the maximum was 83.3%, the minimum was 7.03 %, all have reached significant level.