Function Domain Analysis Of A Pathogen-Inducible Promoter, GST1, In Transgenic Citrus During Xanthomonas Nxonopodis Pv.Citri Infection

Citrus canker is a significant bacterial disease caused by Xanthomonas nxonopodis pv.citri (Xac). There is still no effective method to control this disease. In plant disease resistance breeding, pathogen-inducible promoter has become a mainly strategy to engineer transgenic plants with enhanced and durable disease resistance. The GST1 pathogen-inducible promoter from potato has broad-spectrum response to pathogenic bacteria. The GST1 promoter can efficiently response to both wound-and Xac pathogen-induction in transgenic citrus. So identifying its key cis-acting elements responding to Xac pathogen infection will be benefited for construction of specific expression system for citrus resistant engineering.In this study, the cis-acting of the canker pathogen-inducible promoter GST1 were evaluated in transgenic citrus, and the main results were as follows:1. Bioinformatics analysis of GST1 promoterWe have investigated pathogen-associated cis-acting elements in GST1 promoter. It contains 5 W boxes (TTGACC/T),3 GT1 elements (GAAAAA),12 dofs (AAAG),2 G box variants (CACNTG),1 S box (AGCCACC) and 1 GST1 box.2. Construction of 5’-truncated pathogen-inducible promotersBased on the above results,4 pairs of primers were designed to construct 4 5’-truncated pathogen-inducible promoters by PCR:G1 (1156bp, deleted-1571/-1158), G2 (746bp, deleted-1157/-748), G3 (314bp, deleted-747/-315), and G4 (187bp, deleted-314/-188) promoters.3. Construction of plant expression vectors containing 5’-truncated pathogen-inducible promoterThe four promoters were unloaded from T-vector by HindⅢ and BamH. Ⅰ,and then inserted into HindⅢ/BamH Ⅰ-digested p1300GNGM vector to construct four plant expression vectors:pGl:GUS, pG2:GUS, pG3:GUS and pG4:GUS. In these vectors, the expression of gus gene was regulated by pathogen-inducible promoter.4. Agrobacterium-mediated transformation of Jin Cheng orange and identification of transgenic plantsThe four plant expression vectors were transferred into Jin Cheng citrus by Agrobacterium-mediated method.GFP fluorescence screening, PCR analysis and southern blot hybridization were performed to confirm transformants.36 transgenic plants were obtained in this study.5. wound-inducible characteristics of 5’-truncated pathogen-inducible promotersReal-time quantitative PCR and GUS activity quantitative analysis showed that the pG3:GUS plants can effectively response to wound-induction,while the pGl:GUS, pG2:GUS and pG4:GUS plants can hardly response to it.6. Canker pathogen-inducible characteristics of 5’-truncated pathogen-inducible promotersReal-time quantitative PCR and GUS activity quantitative analysis showed pG3:GUS plants can effectively response to canker pathogen induction, while the pGl:GUS, pG2:GUS and pG4:GUS plants can hardly response to Canker pathogen induction.In conclusion, the fragments from-1571bp to-1158bp and -314bp to-188bp regions contain important pathogen-inducible and wound-inducible cis-elements which can significantly enhanced the activity of GST1 promoter in citrus. The fragment from-1157 to -315 region as a transcriptional repressor might repressed the enhancing functionality of the domains in -314bp to -188bp, and the fragment in -1571bp to-1158bp region may enhance the activity of the GST1 promoter through inhibiting the functionality of the repressor in-1157 to -315 region.