Transgene Studies On Enhancing Pepper Broad-spectrum Disease Resistance By Inducing Hypersensitive Response

Bacterial wilt was caused by Ralstonia solanacearum.The disease development depends on the action of several proteins secreted by the typeâ…¢and typeâ…¡secretion systems.There were several ways to improve disease resistance of crops to Ralstonia solanacearum,such as, transgenic technology,breeding resistant cultivars,biocontrol,and so on.In many cases,the attempts to improve plant resistance to microbial attack using genetic modification approaches have failed.Because constitutive expression of exogenous gene appears to affect plant vigour and yield,interferes with reproduction of the plant.A very promising approach to engineer disease resistance makes use of the plant hypersensitive response(HR).By the introduction of gene related HR under control of a pathogen-inducible promoter might be resulted in broad-spectrum disease resistance.Using pepper(Capsicum annuum L) as material,the effects of different factors on the regeneration of pepper cotyledon explants were investigated.The results showed that the juice of pepper seedlings,AgNO₃,ABA and Sperdine were all important to the differentiation and elongation of adventitious buds.By optimizing the culture conditions,the rate of adventitious bud induction,elongation and rooting reached to 100%,83%,and 100%,respectively.At the base of the highly efficient regeneration system,the transformation condition was optimized.After 2 days of pre-cultivation,the explants were followed by co-cultivation with Agrobacterium tumefaciens for 4 days and delay selection step was canceled.The explants were directly transferred to bud induction medium with Kanamycin 100 mg/L and Cefotaxime Sodium 500 mg/L under continuous light.Of 24 plants,18 were PCR-positive.The result showed that 75%of the regenerated plants were transgenic and that the NPTâ…¡gene was successfully integrated into the pepper genome.According to the sequences of pathogen-inducible plant promoters(PPPs) at GenBank,three promoters were cloned from tobacco genome.They were used to replace cauliflower mosaic virus 35S promoter at pBI121.Following the introduction of each recombinant plasmid into Agrobacterium tumefaciens GV3101,Agrobacterium-mediated transformation of pepper was performed.Positive plants were confirmed by PCR analysis and kanamycin choice.24h after inoculation with Ralstonia solanacearum,GUS activity of transgenic peppers were determined.The results indicated that PPPs could be induced evidently by Ralstonia solanacearum.Amplified from tobacco genomic DNA,PPP3 was cloned into the vector pUC19 and transferred into E.coli DH5α.Then,the putative clones were selected by blue-white cloning qualified and PCR.The result of sequencing was as same as that had been reported.The PPP3 were digested with restriction enzymes Hindâ…¢and BamHâ… .Meanwhile,pflp-pBI and hrap-pBI were digested with the same enzymes.The object DNA fragment were purified and ligated and the ligation products were transferred into the E.coli DH5α.The PCR results showed that the PPP3 were inserted into pBI and ligased with hrap gene or pflp gene correctly.Then,the fragment PPP3+pflp+nos was amplified and inserted into the Hindâ…¢site of PPP3+hrap+nos fragment.At last,the recombined plasmids were transferred into Agrobacterium tumefacie GV3101.PCR detection and sequencing result showed that the vector harboring PPP3+pflp+nos and PPP3+hrap+nos was constructed successfully. The recombined plasmid was used to transformed into peppers by Agrobacterium-mediated transformation.The results of PCR and Southern blotting showed that the pflp and hrap genes were successfully integrated into the pepper genome.The result of reverse transcription polymerase chain reaction(RT-PCR) showed pflp and hrap had partly basal expression.Using actin gene as reference,the transcript efficiency of pflp and hrap was calculated by real-time quantitative PCR.6 hours after inoculation with Ralstonia solanacearum,mRNA of pflp was 13.08 times higher and that of hrap was 9.93 times higher.The transgenic peppers showed obvious hypersensitive response to increase resistance to Ralstonia solanacearum.After inoculation with R.solanacearum,the H₂O₂ content increased 27.4%,the activity of SOD increased 55.2%,the activity of PPO increased 66.0%,the activity of CAT decreased 21.3%,the MDA content decreased 9.2%.Inoculated with spores of Phytophthora capsici,transgenic peppers showed markedly enhancement of disease resistance.The results suggested that under the control of PPP3,pflp and hrap show inducible expression to enhance disease resistance to Ralstonia solanacearum and Phytophthora capsici.Compared with non-transgenic peppers,the chlorophyl content of transgenic peppers decreased to 84.3%.The photosynthesis rates of different transgenic peppers showed apparent difference,and the rates were less than the rate of control.Judged by phenotypes,the growth and development of transgenic peppers were delayed and the area of leaves decreased.It suggested that the integration of pflp and hrap increased the diseases resistance of peppers but there are negative effects on development and photosynthesis.