Studies On Over-expression Of WNK Kinase Genes In Transgenic Rice

Xa21is the first disease resistance gene cloned from rice (Oryza sativa L.), which encodes a spectrum against Xanthomonas oryzae pv. oryzae (Xoo.). Based on yeast two-hybrid technique, it has been identified that XB3can interact with XA21and there may be interaction between the WNK (with no lysine (K)) kinases and XB3proteins. In present studies, a series of specific vectors for over-expression of WNK genes were constructed, all the constructs were introduced into a susceptible japonica rice cultivar Taipei309(TP309). Lots of transgenic rice plants have been generated, and their disease resistances were then investigated. The present results will help us understand the molecular mechanisms of Xa21signaling pathways in rice. The main results are as followings:1. According to the published sequences, the coding region of each WNK genes was identified, their coding sequences were cloned and compared, and also used for construction of over-expression vectors, respectively.2. By Agrobacterium-mediated transformation, all the constructed vectors were introduced into Taipei309, and lots of transgenic rice lines were regenerated. The results from both PCR analysis and Hygromycin resistance test showed that the transgene had integrated into the genome of rice plants. Furthermore, several transgenic lines containing homozygous transgene were selected.3. The total RNAs were extracted for RT-PCR analysis in some transgenic rice plants. The results showed that the expression of endogenous target gene(s) was increased with different ranges in the leaf of transgenic plants containing the transgene. It indicated that the designed structure has functioned in transgenic rice.4. A mixed Xoo bacteria was used for treatment of the transgenic plants as well as their wild type Taipei309. The results showed that the Xoo resistance of transgenic plants had been improved.5. For testing the relationship between Xa21and WNK, the WNK transgenic plants were crossed with transgenic rice with Xa21gene. The selected combined transgenic plants were treatment with P6bacterium. The results showed that there was positive effects of the resistance after combination of two transgenes.