| Banana is a widely favored tropical fruit all over the world. Cultivation of banana and plantain is now threatened by many fungal diseases. Panama wilt in particular, caused by Fusarium oxysporum f. sp. Cubense (race 4) is most serious fungus that has caused dramatic crop damage and economic loss. The genetic engineering of crops has paved a new way for its improvement, but disease resistance gene for banana improvement is limited. Zhongshandajiao (ABB) shows strong resistance to diseases, especially to Panama wilt. However, the mechanism of its resistance to the diseases is still elusive. In this research, we used Zhongshandajiao as a starting material to identify R gene analogues (RGAs) and NPR1 gene. Two parts of studies have been carried out in this thesis.Isolation and analysis of the Resistance Gene Analogues (RGAs) in bananaAccording to the conservative regions of the nucleotide-binding site and the leucine-rich repeat (NBS-LRR) in resistance genes, the polymerase chain reaction with degenerate primers was employed to isolate R gene analogues (RGAs) from five species of banana (Musa spp.), i.e. species of Gongjiao (AA), Xinyiyejiao (BB), Zhongshandajiao (ABB), Fenjiao (AAB) and Taijiao (AAA), respectively. As a result, totally 98 sequences were typical of RGAs out of 208 clones sequenced, of which 33 sequences with identity of deduced amino acid sequence below 97% were further analyzed. Based on the phylogenetic analysis by using MEGA software, the 33 sequences could be divided into 12 distinct MuRGAs. All of which belong to the non-TIR-NBS type. It suggested that the TIR-NBS-type of R genes were selectively lost in banana in evolution process. Comparison and phylogenetic analysis of the MuRGAs with the known R genes from other species revealed their relationship in evolution. Despite the high diversity of the RGAs found in banana, the 12 MuRGAs were detected in all banana species tested, with the exception of MuRGA-I, which did not present in the wild species of Xinyiyejiao, and the cultivated clones of Fenjiao and Taijiao, suggesting there was different NBS sequence between Zhongshandajiao and other cultivated banana. It is the first report that all NBS belong to non-TIR-NBS in banana.Cloning and analysis of SAR related gene MuNPR1-1 in bananaNPR1 is a key regulator of SA-mediated systemic acquired resistance (SAR) in Arabidopsis, that functions at a position downstream of SA accumulation and upstream of SAR gene induction and induced resistance. We cloned the full-length cDNA of MuNPR1-1 gene by homologous cloning and RACE techniques from Zhongshandajiao. The full-length cDNA was 2189bp long and had an ORF that putatively encoded a polypeptide of 574 amino acids, with a predicted molecular weight of 64 kD. The deduced amino acid sequence of MuNPR1-1 had low homology to the other known NPR1 protein. However, they had a relatively high homology at the functional domain. MuNPR1-1 contained the BTB and ankyrin repeat domain that was the molecular basis for NPR1 function. Plant expression vector pCamMuNPR1-1 harboring the MuNPR1-1 gene driven by 35S promoter was constructed and transferred into tobacco var. NC89 via Agrobaterium-mediated method. PCR and RT-PCR analysis indicated that MuNPR1-1 gene was integrated into tobacco genome and expressed. Analysis for transgenic plant is carrying out. |
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