Genetic Transformation Of Carotenoid Biosynthesis Enzyme Gene(PSY) And HIV-1 Gp120 Gene Into Rice

In this paper, using the embryonal callus induced from rice mature embryos as acceptor, the first selecting gene (PSY) in the genetic engineering about plant carotenoid biosynthesis and HIV-1 gene(gp120) were transformed into rice via Agrobacterium-mediated and bombardment respectively. The study based upon improving the nutrition quality of rice and using transgenic rice as bioreactor to produce plant vaccines against HIV. The system of rice callus regeneration was optimized and the factors affecting transformation were also investigated. The resistant plantlets were examined by PCR and PCR-Southern and the result indicated that the target genes have been inserted into rice genome. The main results were summarized as followed.After statistical comparison of the rate of callus induced from seven rice cultivars, Jindao9901 , Jindao5 and Jinxin8 showed to possess preferable callus quality and high frequency of callus induction , and thus were selected for genetic transformation.The rate of callus differentiation under different concentration of 6-BA, NAA and KT were studied. The result showed that the most appropriate concentration of 6-BA , NAA and KT for rice callus differentiation is 4.0mg/L, 0.5 mg/L and 0.5 mg/L respectively. Rice callus cultivated for 30-60 days are suitable as differentiation material. The sensitivity to Hyg concentration varied differently among different cultivars and the suitable concentration is 40~50mg/L. The suitable concentration ofG418 is high to 300mg/L because various cultivars are not sensitive to it.The factors affecting the transformation have been studied and optimized. The results indicated: too high or low of Agrobacterium concentration would reduce the rate of resistant callus markedly and most suitable ODeoo is 0.6 ~ 0.8. The infection time influence on the rate of resistant callus was less and suitable by 15-20 min. 18~22°C co-culture is optimal. Two days dry treatment could improve callus quality and the rate of resistant callus. The study of genetic transformation by particle bombardment indicated: obtaining the most high rate of resistant callus need two times bombardment and bombardment distance is 9cm.Twenty-four resistant plantlets containing PSY gene were obtained and ten of them gave positive PCR reaction. At the same time, nine containing gpl20 gene were obtained and there of them gave positive PCR reaction by detecting of PCR and PCR-Southern.