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Molecular Cloning Two Rice CYP74 Family Genes and Genetic Transforming the HPL Gene in Which

Author: LiQiang
Tutor: WanJianMin
School: Nanjing Agricultural College
Course: Genetics
Keywords: rice (Oryza sative L.) CYP74 cyochrome P450 AOS HPL DES genetic transformation
CLC: S511
Type: Master's thesis
Year: 2005
Downloads: 208
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Abstract


CYP74 is a gene family of plant cytochrome P450 monooxygenases.This family contains AOS (Allene Oxide Synthase), HPL (Hydroperoxide Lyase) and DES (Divinyl Ether Synthase), all of which are enzymes involved in the metabolism processes of plant fatty acids. The catalysates of these enzymes play an important role in many processes of plants such as the responses to development and environments. In this study, we cloned a new rice HPL gene and a AOS gene with PCR-based strategy, and we transformed the rice HPL gene back to rice via Agrobacterium-mediated method. In addition, the characteristic and the distribution in rice genome of CYP74 family members were studied on the genome level.Main results are as follows:1、 Using Bioinformatic analysis combined with PCR-based strategy, we cloned a rice HPL gene (OsHPL1),and constructed two binary expression vectors for genetic transformation: pPZPH2a3(+)-HPL1 and pPZPH2a3(-)-HPL11. The two binary vectors contain full length of sense or antisense rice HPL gene respectively, and both with CaMV 35S as promoter, and hygromycin for selection.2、 Japonica rice Nipponbare was transformed with both pPZPH2a3(+)-HPLl and pPZPH2a3(-)-HPL1. Japonica rice PL2 was transformed with pPZPH2a3(-)-HPL1. Total 77 regenerated shoots were obtained , in which, 64 (83.1%) regenerated shoots were positive with the in vitro leaf assay of hygromycin resistance. PCR analysis and T1 generation assay of hygromycin resistance also showed that foreign genes were integrated into rice genome. Southern blot showed that foreign genes were integrated with more than one copy style in most regenerated shoots.3 、 A new rice AOS gene (OsAOS2) was cloned with bioinformatic analysis combined withPCR based strategy. A prokaryotic expression vector (pET30a-AOS2) was constructed. Prokaryotic expression with E.coli showed that OsAOS2 gene was expressed as inclusion bodies, and the expression amount was positively related to the inducer of IPTG.4、 The distribution of CYP74 family members in rice genome was studied using the method of bioinformatics. BLAST analysis showed that there are five CYP74 members in rice genome. In which, one is HPL gene (CYP74B subfamily), and the other four are AOS genes (CYP74A subfamily). There are no members of CYP74C or CYP74D in rice genome. All five rice CYP74 genes are single exon genes. Sequence analysis showed there are no obvious differences for these five genes between the two main subspecies (Japonica and Indica) of rice. In addition, the expression profile, chromosome position and the upstream control elements of these five rice CYP74 genes were analyzed..

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