Studies On Hypersensitive Response Mediated By TMV Resistance Gene And Cloning Of Homologous Gene In Nicotiana Tabacum

Tobacco is an important economic crop. It is very common phenomenon that tobacco is infected by Tobacco mosaic virus (TMV) and causes mosaic symptom in our country, which affects seriously the yield and quality of tobacco. The most effective method is breeding resistant varieties to prevent and control the disease. Therefore, it is great significance to study hypersensitive response (HR) induction by the interaction between the resistance gene N and TMV, and to clone and characterize a novel resistance gene.In this experiment, the tobacco cultivar Nicotiana tabacum cv. Xanthi was used as material. The time and temperature sensitivity of HR induction by TMV infection, as well as the effects on HR induction and necrotic lesion formation with different concentrations of TMV inoculation were studied. Meanwhile, the resistance gene N homologous gene was cloned, and their main functional domains were analyzed. The results provided the theoretical basis for further understanding the mechanisms of plant disease resistance and breeding tobacco varieties resistant to virus. Main research results were as follows:1. By observing the symptoms of N. tabacum cv. Xanthi NN containing the A gene and N. tabacum cv. Xanthi nn lacking the A gene infected by TMV, the results showed that leaves tissues of lateral veins and branch veins were translucent, and leaves thickness was uneven by TMV infection in Xanthi nn, which was known as mosaic disease. But a clear HR was induced by TMV infection in Xanthi NN, and formed necrotic lesion and produced resistance to TMV.2. By monitoring the time of HR appearance with TMV inoculation, the results indicated that HR began to appear on the site of infection at36hours post TMV inoculation and HR symptom became clear at48hours post TMV inoculation. Subsequently, the cells died gradually and formed necrotic lesion on the site of inoculation. After TMV inoculation, tobacco leaves appeared typical HR at22℃or28℃, but HR did not appear at15℃or35℃. These results demonstrated that HR induction had the time and temperature sensitivity.3. By investigating the HR induction by infection with different concentrations of TMV, the results showed that no matter what the concentration of TMV extract was, HR began to appear at36hours post TMV inoculation at22℃and HR began to appear at48hours post TMV inoculation at28℃. These were accordant with the preceding result, and illuminated that the effect of TMV concentration on HR induction was not obvious.4. By studying the size and density of necrotic lesions by infection with different concentrations of TMV, the results showed that when tobacco leaves were inoculated with different concentrations of TMV, whether at22℃or at28℃, TMV concentration effected on the sizes of necrotic lesions, namely the sizes of necrotic lesions increased at high concentration and changed little at low concentration, and the density of necrotic lesions also had certain correlation with the concentration.5. According to the known sequences of tobacco N gene, specific primers were designed, and a cDNA fragment of N homologous gene (named NH1) was cloned by RT-PCR. Sequence analysis indicated that NH1cDNA contained2040bp nucleotides, and encoded679amino acids. Comparision of amino acids sequences discovered that NH1had higher homology with other Nhomologous genes. Structural analysis showed that NH1included P-loop, Kinase-2, Kinase3a, hydrophobic domain (HD), and3typical leucine-rich repeat domains (LRR), which belonged to the NBS-LRR class protein.6. NH1gene are expressed in Xanthi NN.Tobacco leaves infected by TMV for0h,12h,24h,36h,48h,60h,72h at22℃were selected to extract RNA, and the expressions of the NH1gene were detected in different time by semi-quantitative RT-PCR The results showed that the expressions of NH1gene were clear at0h12h,24h,36h and48h post TMV infection, but the expressions of NH1gene descend slightly at60h and72h post TMV inoculation. These demonstrated that TMV inoculation did not affect the expression of NH1, but the necrotic lesion and cell death in leaves could affect the expression of NH1.