Effect Of The Presumed Signal Peptide Sequence Of HpaXm On Its Expression And Action Sites In Transgenic Tobaccos

Harpin, encoded by hrp (hypersensitive response and pathogenicity) gene of gram-negative plant pathogens, is a kind of protein Elicitors. It can stimulate HR (hypersensitive response) reaction in non-host plants. HpaXm was identified from Xanthomonas citri subsp. Malvacearum (a synonym of Xanthomonas campestris pv. Malvacearum (Smith1901) Dye1978, Xcm). It also locates in the hrp region and shares the common characteristics of harpin. Previous researchers have shown that exogenous administration of hpaXm can induce plants producing HR (hypersensitive response, HR), and enhance tobaccos resistance to TMV. Additionally signal intellectual property (IP) prediction suggested that a putative signal peptide of hpaXm occurred on the N-terminal leader peptide (1-MNSLNTQIGANSSFL-15).In this paper, we studied the hpaXm and the N-terminal leader peptide of hpaXm via transgenic tobaccos. The first part described development of transgenic tobaccos (Nicotiana tobacum cv.Xanthi nc) expressing The full-length HpaXm and the signal peptide like segment-deleted mutant HpaXmALP from Xanthomonas citri subsp.malvacearum, with the method of Agrobacterium-mediated transformation. The second part described extracted soluble protein activity assay and the transgenic tobaccos’resistance to TMV assay.The results showed that transgenic tobaccos were obtained by molecular identification. Detection of the target gene and the35S promoter sequences by PCR and the target genes sequences by PCR-Southern blot hybridization in genome from the T1line positive transgenic plants, showed that the hpaXm gene and hpaXmALP gene had already integrated into the recipient genomes respectively. Reverse transcriptase PCR indicated that the transferred genes expressed normally. Soluble proteins extracted from plants transformed with hapXm and hpaXmALP could stimulate hypersensitive response on tobacco leaves respectively, indicating that hapXm or hpaXmΔLP expressed in transgenic tobacco plants are bioactive. Disease resistance bioassays showed that, the tobacco plants transformed with HpaXm gene enhanced resistance to TMV similarly as those transformed with hpaXmΔLP gene. It suggests that the signal peptide like fragment is superfluous for the endogenous expression and activity of hpaXm, and ability of hpaXm inducing tobacco resistance against TMV. Immune colloidal gold detection showed hpaXm expressed in transgenic tobaccos can be transferred to the cell membrane and organelles and even to the cell wall of tobacco, but the presumed signal peptide sequence deletion mutant hpaXmΔLP expressed in transgenic tobaccos can not cross the membrane to reach the cell wall. In summary, the N-terminal amino acids before1-15hpaXmΔLP deletion does not affect the expression in transgenic tobaccos, but it can prevent hpaXmΔLP being transferred to the cell wall, so as to affect the disease resistance of transgenic tobaccos. Even though there was no significant difference between the two groups of transgenic tobaccos.