Canker Bacterium-and Wound-response Characteristics Of Three Pathogen-induced Promoters In Transgenic Citrus

Using constitutive promoter such as CaMV35S regulating the expression of resistance gene is the common expression strategy in plant disease resistance genetic engineering breeding. This strategy usually results in the overaccumulation of heterologous protein and metabolite products in plant, breaking intrinsic metabolite balance and having reverse effect on the output and quality. Some metabolites are not indispensable even more have toxic action to plant, so hinder plant normal grow even result in dead. The expression of resistance gene under control of an Appropriate pathogen-inducible promoter could avoid foreign gene excessive expression in plant, and then decrease or eliminate negative effects to plant growth and development. There is little research report about using pathogen inducible promoter regulate the expression of resistance gene in citrus andno more research literature related to pathogen inducible promoter derived from citrus has reported. So how to use pathogen inducible promoter from other species for improving resistance to citrus canker deserve study.In this study, The characteristic of pathogen inducible promoters from tobacco and potato were evaluated in transgenic citrus, the main result as followers:1. Cloning of pathogen inducible promoterthe pppl and hrs203j pathogen inducible promoters, which response to various pathogenic bacteria, derive from tobacco; the gstl promoter from potato also responses to broad-spectrum pathogenic bacteria.we design three pair primers according to the base sequence of pppl,gstl and hrs203j promoters. PCR amplification and T-clone sequencing obtain correct promoter sequences. 2, Construct of plant expression vectorThe three promoters were unloaded from T-vector using HindⅢ and BamHI, and then inserted into HindⅢ/BamHI digested pBI121vector to construct these plant expression vectors:PBI121-ppp1、PBI121-gst1、PBI121-hrs203j. In these vectors, the expression of gus gene were regulated by pathogen-inducible promoter.3, wound-inducible characteristics of three promoters1) GUS histochemical staining results showed pppl and hrs203j promoters kept silence before wound-inducible, while gstl have background expression. pppl promoter showed the highest activity level after24hs of wound-inducible,meanwhile, gstl promoter showed the strongest activity after12hs of wound-induction; hrs203j is hardly inducibled by wound.2) Real-time PCR and GUS enzyme activity quantitative analysis showed that the expression of gus gene driven by pppl promoter is very low before wound-induction, but is Significantly enhanced after24hs of wound-induction.The background avtivity of GUS is detected before gstl promoter is induction, after12hs of wound-induction, remarkedly increased GUS expression are found.4, Canker bacterium-inducible characteristics of three promoters1) GUS histochemical staining results showed no blue stain was detected in both pppl and hrs203j transgenic citrus, but in gstl transgenic citrus background blue stain could be detected before Canker bacterium induced. Blue spots were observed at the infection sites in leaves from Pppl transgenic citrus in five days of culture when they were infected by Canker bacterium.After ten days, strong blue spots were detected at the infection sites in pppl and gstl transgenic leaves.2) Real-time PCR and GUS enzyme activity quantitative analysis showed pppl promoter driving gus gene expression is very low without Canker bacterium infection, then gus gene expression was Significantly enhanced after ten days of Canker bacterium induction. gstl promoter showed background activity before Canker bacterium induced, then gus gene expression was remarkedly enhanced after ten days of Canker bacterium induction.