Study On The Transgenic Tobacco Expressing Harpin_Xoo And Its Two Deletion Derivatives

Most gram-negative bacterial pathogen possess hrp (hypersensitive response and pathogenicity) gene cluster, which controls the ability of the pathogen to cause disease in susceptible plants and elicit HR in resistant plants. Harpins, a group of proteins encoded by hrp genes of gram-negative bacteria, can elicit hypersensitive cell death in tobacco leaves, induce disease and insect resistance in plants, and promote plant growth. hrfA (HR function) gene was identified by our lab in a Japanese strain of Xanthomonas oryzae pv.oryzae. It is 420 bp in length and encodes harpin_xoo protein, which can also elicit HR in tobacco and induce disease to many pathogens. Harpin_xoo has some specialties: rich in Gly, it also contains Cys; moreover, it is smaller than many other harpins. Our lab has successfully transformed hrfA into tobacco, rice and cotton, and disease resistance of these transgenic plants have been enhanced. Li Ping has cloned five deletion derivatives of hrfA (i.e. AB, CD, EF, AD and CF) by PCR and expressed them in Escherichia coli BL21/pET30a(+). Fragment AB, which contains N-terminal 59 amino acids, can trigger HR and induce resistance and is the best one with multifunction among deletion derivatives. The middle fragment CD (99 bp) fails to trigger HR but can induce resistance normally. When combined with other fragments, CD can enhance their bioactivity, thus, the fragment CD may act as enhancer in triggering HR. So it would be very interesting to study the resistance mechanisms of AB and CD.In this study, we excised the hrfA gene and its two deletion derivatives, AB and CD, from pET30(a) by digestion with restriction enzymes BamH I and Xbal I, and inserted them into the transformation vector pBI121 between the CaMV35S promoter and theβ-glucuronidase gene uidA. The resulting three recombinant constructs, pB-hrf, pB-AB, pB-CD, were transformed to E. coli DH5a. After verifying correct orientation of genes, these three transgenic consrtucts were transformed into Agribacterium tumefaciens EHA105 by the freeze-thaw method. Transformation was conducted by infection of tobacco leaf discs with recombinant A. tumefaciens, and transgenic tobacco plants were obtained by screening with kanamycin. Genomic integration and expression of the transferred genes were determined by PCR, PCR-Southern blot hybridization and reverse transcriptase PCR. Soluble proteins extracted from plants transformed with hrfA and AB could elicit HR on tobacco leaves, but those from CD-transformed plants could not.Under the same culture conditions, there was no apparent difference between the transgenic and parent plants in heights, size of leaves, and flowering time. The transgenic plants growed normally, without macroscopical HR. When stained with trypan blue, leaves from plants transformed with hrfA and AB showed micro-HR, whereas leaves from CD-transformed plants could not. Disease bioassays showed that, the tobacco plants transformed with deletion derivatives, AB and CD, enhanced resistance to TMV similarly as those transformed with the whole hrfA gene.