The Construction Of Monocotyledon Expression Vector With Coptis Japonica 4-hydroxyphenylpyruvate Dioxygenase Gene And The Genetic Transformation Of Rice

ρ-hydroxyphenylpyruvate dioxygenase (HPPD) is an enzyme which needed in the second step of the tyrosine catabolic pathway. It catalyzes the formation of homogentisate from p-hydroxyphenylpyruvate. In plants, homogentisate is the precursor of vitamin E and plastoquinones, therefore HPPD also plays a key role in the biosynthesis of this two kinds of substances. Three types of herbicide families, isoxazoles, triketones and pyroxazoles, take HPPD as the target site. They are the inhibitors of HPPD activity, and result in the bleaching and death of plants. At the same time, the herbicide-resistant transgenic crops had also aroused people's widespread interest and the attention. It was found that Coptis japonica HPPD had certain resistance to the herbicide DTP.The rice (Oryza saliva L.) which as the pattern plant of genetic transformation of the monocotyledon has laid the foundation for the gene transformation and the gene regulation research of other gramineae crops. The developments research on rice gene rapidly in the near several years, some type of rice has been completed the entire genome sequencing and has obtained many transgenic rice which stable heredity through the transgenic technology. The people attempt to study the foreign gene expression in the monocotyledon pattern plant through transfered some useful genes to rice.In this study, we constructed an expression vector with herbicide-resistant gene Cjhppd and transferred this gene to rice mediated by Agrobacterium tumefaciens. PCR analysis showed that the Cjhppd gene had already integrated into rice cells. The main experimental process was summarized as follows.1.Construction of the monocotyledon expression vector which included cjhppd, Ubi and Npt.2.Rice embryogenic calli was infected by EHA105. We successfully obtained 76 resistanct calli and the resistant callus rate was 40.2%, and obtained 33 regeneration plants which the resistant adult regeneration rate was 17.5%.3.We successful obtained transgenic (Cjhppd gene) rice, and this was confirmed by PCR analysis. The positive transgenic rice with Cjhppd gene was 8 of 33.4.Analysis of resistance of herbicide DTP showed that the leave of wild type rice became more bleaching than that of the transgenic type in the presence of DTP. Chlorophyll a, chlorophyll b and carotenoid contents in the transgenic plant are higher than that of the wild type under DTP treated condition. It illustrated that overexpression of HPPD could enhance the resistance to herbicide DTP of transgenic rice.