Genetic Transformation And Promoter Activity Assays Of Plant Safety Expression Vectors Harboring Two Tandem Pathogen-Inducible Promoters

Complex infections challenge the genetic engineering of plant disease resistance. Constitutive over-expression of defense components frequently results in poor quality of plants in the genetic engineering of plant disease resistance. Use of pathogen-inducible promoters has advantages than constitutive promoters in genetic engineering of disease resistance. Single pathogen-inducible promoter can supply transgenic plant a limited resistance spectrums to pathogens. If using two tandem pathogen-inducible promoters, it maybe gives transgenic plant wide resistance spectrums of pathogens.To test whether two tandem pathogen-inducible promoters can enlarge the resistance spectrums of plant to pathogens, we have constructed four plant safety expression vectors based on p2301NG, which backborn is pCAMBIA2301. These vectors harbored two T-DNAs and the selective marker gene NPTII expression cassette was separated in different T-DNAs from the GUS expression cassette driven by two tandem highly pathogen-specific inducible promoters. The main results and conclusions are as follows.1 Construction and Genetic Transformation of Plant Safety Expression VectorWe had constructed four plant safety expression vectors based on p2301NG. EAS4 and hsr203J, were used to drive report gene uidA. Consideration of the possible position affection of EAS4 and hsr203J, we had constructed two recombinant plasmids HP1+EP2 (hsr203J+EAS4+uidA) and EP1+HP2(EAS4+hsr203J +uidA). Two other vectors HP1 (hsr203J+ uidA) and EP1 (EAS4 +uidA) harboring each promoter were used as control. 200 regenerating plants were obtained by transforming tobacco using these constructs. 152 of the regenerating plants were transgenic plants after detection with PCR. 2 There are obvious inducible GUS activity in transgenic tobacco and the GUS activity in tobacco transformed with vectors harboring two tandem promoters were significantly stronger than that of single promoter.We have tested the inducible activity of different promoters in transgenic tobaccos with RT-PCR, histochemical staining and fluorescence quantitative assays.2.1 Thirty transgenic tobacco plants were treated with Parasiticein for 6h. Expression of GUS gene at the transcription level was detected via RT-PCR in 24 transgenic plants.2.2 We selected randomly 14 of the 24 transgenic tobacco plants for further analysis. Induction with Parasiticein and inoculation of RALSTONIA solanacearum and the spore suspensions of Phytophthora nicotianea[0] and Botrytis cinerea were carried out. Histochemical staining showed that GUS activity were detected in all of the 14 transgenic tobacco plants and only the transgenic tobacco plants caring ESA4 promoter were not inducible by B. cinerea, showing that all the vectors we constructed can work and the tandem two promoters are able to enlarge resistance spectrums of pathogens.2.3 Quantitative differences between two tandem promoters and single promoter were analyzed by fluorescence quantitative assays of GUS activity. Transgenic plants were induced with Parasiticein or inoculated with R. solanacearum and spore suspensions of P. nicotianea[0]. GUS activity in the tobacco plants transformed with the construct HP1+EP2 was 5.9-8.0 times higher than that transformed with the construct HP1, and 6.7-7.9 times than EP1. GUS activity in tobacco plants transformed with the construct EP1+HP2 was 5.0-5.7 times higher than that transformed with the construct HP1, and 4.7-6.5 times than EP1. There is no significant difference between HP1 and EP1. 2.4 Analysis of GUS activity using fluorescence quantitative assay showed that the peak value of the GUS activities in tobaccos transformed with the construct HP1+EP2 appeared from 6 to 10 hours after being induced, while the peak value of the GUS activities in tobacco plants transformed with the construct EP1+HP2 can be observed from 8 to 14 hours after being induced. GUS activities in transgenic tobacco plants harboring two tandem promoters were always higher than that in tobacco plants transformed with the construct harboring single promoter.Our investigation showed that the four plant expression vectors we had constructed can work well in transgenic tobacco plants and the two tandem promoters can not only enlarge resistance spectrums of pathogens but also enhance inducible GUS activity.3 It was verified that the selective marker gene NPTII was separable from report gene uidA in the self-crossing progeny of transgenic tobacco plants.Elimination of selective marker genes is a very important part for production of the edibly safe and enviroment-friendly transgenic plants. The four vectors we had constructed separated the selective marker gene NPTII expression cassette from the GUS expression cassette in twin different T-DNAs. We can gain the transgenic plants without NPTII gene but only holding uidA gene after self-crossing of the transgenic plants.The separation ratio of NPTII gene in self-crossing progeny T1 generations of the 13 lines of transgenic plants is 2.89:1, indicating the NPTII gene was integrated into the genome of the transgenic plants with single copy and can inherit following the Mendel'genetic law.To test whether NPTII gene is independent to uidA gene, we detected them by PCR. Detection of the transgenic plants showed transgenic plants only with NPTII gene account for 22.31%, transgenic plants only with uidA gene accounts for 20.77%, transgenic plants holding these two genes account for 53.85%. The separation ratio in self-crossing progeny T1 generation accords with the genetic law of independent assortment by X2 analysis.