| During the process of plant growth and development, plants often suffer biotic or abiotic stress, but plants have developed a set of stress-response mechanism in the long-term evolutionary process. There is a certain type of transcriptional regulation factor AP2/ERF (Apetala2/enthylene response factor), which plays an important role in regulating plant response to biotic or abiotic stress. Research showed that AP2/ERF transcription factors played a regulation role in signal transduction pathways of plants sufferring viral invasion and causing defense reaction to acquire resistance. For the signal pathway of the interaction between Tobacco Mosaic virus resistance gene N with TMV to induce HR, it is not still clear that what transcription factors take part in and how to play a regulation role in HR induction.In this experiment, a tobacco cultivar Nicotiana tabacum cv. Xanthi NN was used as material. The regulation role of ERF during the signal transduction pathway of the interaction between the N gene and TMV to induce HR was studied. The results provided some theoretical basis for further understanding the signal pathway of HR induction and the molecular mechanism of the interation between the N gene and TMV. The main results were as follows:(1) When tobacco was inoculated with TMV, a local HR began to appear at around36h after TMV inoculation, but a clear HR was induced at48h. With the time of inoculation going on, HR was strengthened, and the sizes and numbers of necrotic lesions increased gradually. Until72h, the HR spread to all inoculated leaves.(2) The target fragment was amplified by RT-PCR from Xanthi NN leaves inoculated with TMV at48h, and the size was422bp.3’,5’cDNA fragments were592bp and435bp by3’and5’RACE, respectively. The full-length NtERF1-1cDNA was obtained by matching, which contained1091bp nucleotides including690bp ORF, and encoded229amino acid residues.(3) Amino acid sequences analyses showed that NtERF1-1had more than95%homology to NtERF6b and NbCDl in Nicotiana, and did not contain LDxxP motif, belonging to the transcription activating factors. Predicted molecular weight of NtERF1-1protein was24.58kDa, and average hydrophobicity was-0.474, and the isoelectric point was pH9.90. Meanwhile, the secondary structure was calculated, and NtERF1-1was localizated in the nucleus without transmembrane domain, and there were many modification sites of related proteins. (4) RT-PCR and qRT-PCR analyses showed that the expression level of NtERF1-1was low without TMV inoculation, but the expression level increased gradually after TMV infection. Until48h after TMV inoculation, the expression level reached the highest value, and then the expression level reduced a little. The results indicated that the NtERF1-1gene was induced by TMV.(5) Some related signal molecules were tested in tobacco leaves inoculated with TMV, the results showed that H2O2accumulated slowly from0to24h, and increased rapidly from24to36h, until around48h reached peak (28.94μmol·g-1), then decreased. The content of SA increased up to the maximum (0.713203mg·g-1fw) at around48h, then took a downtrend, and the content of SA was only0.113198mg·g-1FW at Oh after TMV inoculation, which was1/6of the content at48h after inoculation. These results demonstrated that the contents of signal molecules H2O2and SA increased gradually along with TMV infection, and reached top levels when the HR was induced.(6) The expression model of NtERF1-1was analyzed by applying exogenous SA, Eth and JA. The results indicated that for SA treatment group, the expression of NtERF1-1rised gradually from12to24h, and reached the maximum at24h, then remained at a certain level. For JA treatment group, the expression of NtERF1-1increased at24h, and reached the highest level at around48h. When treatment with Eth, the expression of NtERF1-1began to increase at24h, and reached the maximum at36h, then hold at a certain level. These results demonstrated that SA, JA and Eth could facilitate the expression of NtERF1-1. |
PDF Full Text Request