Selection Of Reference Genes In Citrus And Expression Of PthA Gene In Transgenic Sweet Orange

Citrus canker disease caused by Xanthomonas axonopodis pv.citri.affects almost all of citrus species and cultivars in the world and has caused severe damages to most citrus producing areas worldwide. In the previous research, pthA gene was cloned into a plant expression vector system and transferred into sweet orange via Agrobacterium-mediated transformation. The object of this study is to provide a comprehensive molecular identification of all transgenic clones for clear the integration of the target gene and its copy numbers. And then, analyzing the expression status of target genes when plant under normal circumstances by quantitative-PCR, as well as after pathogen inoculation. Thus, Providing the basis to explore the relationship between pthA-nls gene expression levels and degree of plant resistance to citrus canker. The main results were as follows:1. The pthA-nls gene and pthA-a-nls gene was integrated into the succari orange (T1-T6, Ta1-Ta9) and Bingtang sweet orange (B3-B8) genome by PCR and Southern blot, respectively. Southern blot results revealed that one copy was inserted to T1,T2,T4,B3,B4,B5,B6,B7,Ta5,Ta8 transgenic sweet orange clones, two and three copies were inserted to T3,T6,B8 and T5 transgenic sweet orange clones, respectively. RT-PCR showed that two target genes were successfully expressed in all transgenic sweet orange clones.2. An effective quantitative PCR technique was established for gene expression analysis in citrus. Three Housekeeping genes(18srRNA, ACTB and rpll) screened out of a large number of housekeeping genes can be used as reference gene in citrus for these three genes proved to be stablely expressed both in six different genetypes and five different tissues. Of these three reference genes for data normalization, will help to get more reliable results.3. All pthA-nls gene transformed sweet orange and parts of pthA-a-nls gene transformed succari sweet orange (Ta5, Ta8) were analyzed by quantitative PCR, the expression levels of target gene in different transgenic sweet orange clones were cleared. 4. The transgenic clones of sweet orange were in vivo tested for disease resistance to citrus canker disease. Parts of transgenic pthA-nls gene clones showed significantly less lesion development than the controls. Quantitative PCR results showed that the expression levels of target gene in parts of transgenic sweet orange clones (T1, T2, T3, T5) increased significantly.