【作者】 李胜军；

【导师】 裴新梧；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 香蕉是重要的粮食作物,也是倍受人们喜爱的热带水果,但由于病虫害,尤其是枯萎病的发生,对香蕉产业造成了巨大损失。基因工程技术的发展为作物改良提供了有效途径,但目前用于香蕉抗枯萎病基因工程的基因资源还非常有限,对香蕉抗病分子机理的研究还较少。鉴于中山大蕉抗逆性强,尤其高抗枯萎病4号小种,本研究利用同源序列法克隆NBS类抗病基因和系统获得抗性途径中的主要抗病信号元件MuNPR1-1基因,取得如下结果:1.中山大蕉NBS类RGAs的克隆与分析根据NBS类抗病基因的保守结构设计简并引物,以中山大蕉基因组DNA为模板,PCR扩增获得了98条NBS序列,进化树分析将其分为12类,与已知的20种NBS类抗病基因的NBS区的进化树结果显示,这12类MuRGAs分布在5个不同的区域,其中的nT subclass 2区域只包括了7类MuRGAs,这可能是不同于其它作物中的一类新的抗病基因。利用RT-PCR检测了12类MuRGAs在不同基因型香蕉中的表达,发现其中的10类均在转录水平表达,但有两类明显不同,MuRGA-I只存在于抗病的中山大蕉和贡蕉基因组中并表达,MuRGA-K只存在于中山大蕉的基因组中并表达,而且这两段序列在进化树的nT subclass 2区域,这揭示了在抗病的中山大蕉和贡蕉中存在着与不抗病的栽培香蕉中不同的NBS类序列,可能与抗病性相关。在对香蕉中NBS-LRR的研究中,发现了不同基因型的中山大蕉(ABB)、贡蕉(AA)、信宜野蕉(BB)、粉蕉(AAB)、泰蕉(AAA)的NBS都属于non-TIR-NBS,首次揭示了香蕉中所有NBS结构均为non-TIR-NBS类。2.中山大蕉MuNPR1-1基因的克隆与分析NPR1是SAR途径中的中心元件,其功能定位在SA积累之后、SAR基因表达之前。利用同源克隆法和RACE技术获得了中山大蕉的MuNPR1-1基因,该基因DNA全长4349bp,有4个外显子,3个内含子,cDNA全长2189bp,阅读框为1725bp,推定的编码蛋白为574个氨基酸,分子量大小为64kD。推定的MuNPR1-1蛋白与已知NPR1基因的氨基酸相似性低于42%,与玉米NPR1的氨基酸相似性为69%。NCBI中rpsBLAST搜索发现MuNPR1-1推定的氨基酸含有BTB/POZ和锚蛋白重复序列,命名为MuNPR1-1 (GenBank登录号:EU7478883)。构建了35S组成型启动子驱动的植物表达载体pCamMuNPR1-1,利用农杆菌转化法转入烟草品种NC89,获得了40个转基因植株,PCR和PT-PCR表明,MuNPR1-1基因已整合到烟草基因组中并能够表达。更多还原

【Abstract】 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.更多还原