【作者】 李康；

【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， 2010， 硕士

【摘要】 香蕉是人们喜爱的热带水果,也是非洲等地的主要粮食作物。但长久以来枯萎病、尤其是枯萎病4号小种,严重影响香蕉生产,造成了大量损失。目前尚无理想的防治药剂,迫切需要培育抗枯萎病的香蕉品种,但是栽培香蕉多为三倍体,难以通过有性杂交的方式改良,植物抗病基因工程为改良香蕉对枯萎病的抗性提供了新的途径。SGT1基因是调控植物抗病反应的一个重要元件,其本身对病原物并无抑制作用,但在植物多种R基因介导的对细菌、真菌和病毒等抗性反应中起着重要作用。本项研究选用抗逆性强,尤其是高抗香蕉枯萎病的中山大蕉,克隆了其中的SGT1基因和病程相关蛋白基因PR-1,获得了以下结果:利用同源序列法克隆了中山大蕉中的MeSGT1基因,该基因cDNA全长为1438bp,5’非翻译区为137bp,开放阅读框为1119bp,3’非翻译区为182bp,该基因没有内含子。推测的编码蛋白为372个氨基酸,分子量大小41645.03 Da,含有三个保守结构域:TRP (tetratricopeptide repeats)、CS(CHORD-containing protein and SGT1)和SGS (SGT1-specific domain),命名为MeSGT1(GenBank:GU591491,2010年3月)。克隆了中山大蕉中的病程相关蛋白基因MePR-1 (GenBank:GU591492,2010年3月),开放阅读框为492bp,编码163个氨基酸。利用RT-PCR的方法分析了MeSGT1、MuNPR1-1和MePR-1基因对香蕉枯萎病4号小种(FOC 4)的应答反应,结果表明:抗病的中山大蕉幼苗叶片经FOC 4接菌诱导后,MeSGT1基因的表达水平在诱导后12h达到最高,高表达持续到24h,在72h降到接近起始状态; MuNPR1-1在诱导后的表达水平与MeSGT1基因同步,病程相关蛋白MePR-1基因的表达在24 h达到最高,48h开始下降,72h又恢复到起始水平。而感病粉蕉中的MeSGT1和MuNPR1-1在0、12和24h无明显变化,48h开始增加并在72h达到最高值,病程相关蛋白MePR-1基因的表达在0、12、24 h无明显变化,在48-72 h有增加。这表明了抗病的中山大蕉的MeSGT1和MePR-1基因对病原菌信号分子的反应比感病品种粉蕉更加敏感,有助于激活下游防卫基因的表达。构建了35S启动子驱动的植物表达载体pCam-MeSGT1,利用农杆菌介导的方法转入烟草NC89,为进一步的功能分析打下了基础。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, of which the fungal disease, Panama wilt in particular, caused by Fusarium oxysporum f. sp. Cubense.race 4 (FOC4) is most serious that caused dramatic crop damage and economic loss. As almost all banana and plantain cultivars are highly susceptible to race 4 of this pathogen and the chemical and cultural control measures are not efficient, the creation of new banana varieties resistant to this disease is crucial. Cross breeding program is difficult to be conducted due to the predominantly asexual behavior of edible bananas, which are triploid and sterile. With the development and optimization of genetic transformation protocol in banana and the discovery of more genes resistant to pathogens, the genetic engineering of banana has paved a new way for its improvement.In plants, SGT1 positively regulates disease resistance conferred by many Resistance (R) proteins and developmental responses to the phytohormone, auxin. In this research, we use Zhongshandajiao which has a high resistant ability, especially to Panama wilt disease as a starting material. The results are as follow:We cloned the full-length cDNA of MeSGT1gene and MePR1 using homologous cloning and RACE techniques from Zhongshandajiao. The full-length cDNA was 1438bp long, the 5’-untranslated region (5’ -UTR)is 137bp,the 3’-untranslated region (3’ -UTR)is 182bp, and an ORF that putatively encoded a polypeptide of 372 amino acids, with a predicted molecular weight of 41645.03 Da. It had a relatively high homology at the functional domain. MeSGT1 contained the TRP (tetratricopeptide repeats), CS(CHORD-containing protein and SGT1)and SGS (SGT1-specific domain) that was the molecular basis for MeSGT1 function(Genbank:GU591491,March 2010)。MePR1 encoded a polypeptide of 163 amino acids(GU591492, March 2010)。RT-PCR is used to analyze the expression changes of MeSGT1 ,MuNPR1-1 and MePR-1 in zhongshandajiao after FOC 4 treatment, The results show that MeSGT1 the expression level reached the highest point in 12h and MePR-1 in 24h after FOC 4 treatment, when it is 72h after FOC 4 treatment, the expression level is just as non-treated.The expression level of MuNPR1-1 show synchronization with MeSGT1 after FOC 4 treatment. It indicates that zhongshandajiao MeSGT1 is sensitive to FOC 4 treatment, and it will activate the expression of defensive genes downstream. However, The MeSGT1 and MuNPR1 -1 from susceptible-Fenjiao have no distinct changes until 48 h after treatment, and the highest level last to 72h.The MePR-1 from Fenjiao show the similar changes,it have no obvious increase before 48h and its peak level take place on 72 h after inducing treatment.It indicated that the MeSGT1 and MePR-1 of resistant-Zhongshandajiao show more sensitive to pathogen signal molecules than that of susceptible-Fenjiao.This sensitivity to pathogen may be favorable to express downstream plant defense genes. Plant expression vector pCamMeSGT1 was constructed and transferred into tobacco var. NC89 via Agrobaterium-mediated method. Analysis for transgenic plant is carrying out.更多还原