【作者】 张兆顺；

【导师】 廖玉才； 李和平；

【作者基本信息】 华中农业大学 ， 作物生物技术， 2008， 硕士

【摘要】 植物抗病性及其机制研究一直是植物病理学和抗病遗传育种研究的热点之一。β-1,3-葡聚糖酶是一种重要的病程相关蛋白,能够降解β-1,3-葡聚糖(大多数病原真菌细胞壁的主要成分),从而使真菌细胞壁破裂,细胞内含物外溢,导致病原菌死亡。体外抑菌试验表明:该酶具有抗菌功效,它不但能够抑制真菌菌丝的生长,而且还可以释放真菌细胞壁的诱导物,从而间接促进植物体内植保素的积累,增加抗病能力,因此该酶在植物抗病工程中具有极高的应用价值。小麦是我国重要的粮食作物之一,长期以来,真菌病害一直是危害小麦生长、影响小麦产量的主要原因之一,给我国的小麦生产带来巨大的经济损失,因此防治小麦真菌病害一直是人们非常关注的问题。本研究根据已知的小麦β-1,3-葡聚糖酶基因(Glu基因)的序列,从小麦的cDNA中通过RT-PCR法克隆获得Glu基因编码序列。将Glu基因构建到原核表达载体,进行原核表达并对所表达的蛋白进行酶活测定和抗真菌的功能鉴定。构建C端带有GFP标签的Glu基因的双子叶植物表达载体,并成功得到转基因拟南芥。分析了Glu基因在小麦品种中国春中不同发育时期,不同组织的表达模式。对小麦中国春基因组中Glu基因的拷贝数进行了研究。结果表明,从小麦的cDNA中分离克隆的Glu基因,在pGEX表达载体和BL21trxB(DE3)宿主菌的原核表达系统中,对Glu基因的开放阅读框片段进行表达,经IPTG诱导表达的谷光甘肽-葡聚糖酶融合蛋白的分子量约为63kD,推算出Glu基因表达蛋白分子量37kD。以昆布多糖为底物的酶活测定证明,在细菌中表达的葡聚糖酶具有典型的β-1,3-葡聚糖酶活性;体外抑菌实验表明,该酶可有效抑制立枯丝核菌(Rhizoctonia solani)菌丝生长。通过Northern杂交和半定量RT-PCR分析表明,Glu基因在小麦中国春中不同时期的不同组织差异表达,开花前后麦穗和叶片中表达量差异较大;芽期接种真菌表明,禾谷镰刀菌(Fusarium graminearum)和大丽轮枝菌(Verticillium dahliae)的侵染诱导Glu基因表达。Glu基因是一个较小的多基因家族,在Southern杂交中检测到4-6个拷贝。这些研究结果为深入研究Glu基因的抑菌机理及其在植物基因工程中的有效应用提供了重要资料。更多还原

【Abstract】 Hydrolytic enzymes such asβ-1,3-glucanases are considered to constitute part of the general array of defense genes induced by pathogen infection in higher plants.Wheat is one of the important crops in China.Fungal diseases have been one of the principal causes of damaging wheat growth and wheat yield losses,and thus effective control of fungal diseases has been one of the concerned questions by the scientists.Since human started to cultivate wheat plants,a lot of measures have been applied to control the fungal diseases,such as breeding the fungus-resistant cultivars by traditional breeding ways,the application of pesticides,and colligated ways to reduce dosage of agrochemicals and rudimental agrochemicals.But due to the limited genetic germplasm resources,the high variability of fungi and the pollution of the pesticides etc,the technology of gene engineering to breed resistant cultivars becomes one of the recent research focuses of agricultural biological technology.In this study,aβ-1,3-glucanase gene(Glu) was amplified by RT-PCR and subcloned into an expression vector pGEX,to construct pGEX-Glu for bacterial expression.A SDS-PAGE analysis indicated that the clonedβ-1,3-glucanase gene was highly expressed in bacteria after induction by IPTG.Enzymetic assays confirmed that the glucanse displayed a clearβ-1,3-glucanase activity.In vitro antifungal assay revealed that the glucanase showed s strong inhibitory activity towards a pathogenic fungus Rhizoctonia solani.The expression pattern of Glu was studied by Northern blot analysis.The results revealed that the glucanase gene expression was developmentally regulated and the Glu transcripts were detectable in both vegetative and reproductive tissues such as spikes and stems,with peaks in spikes one week before floweing.The lowest expression level was observed in spikes one week after floweing.Infection of wheat seedlings by Fusarium graminearum and Verticillium dahliae induced the Glu gene expression.There would be 4 to 6 copies of the Glu gene in wheat genome revealed in Southern blot analysis.The Glu gene and GFP gene were fused together with Glu in the N-terminus and subcloned into plant expression vector pTR.The pTR-Glu-GFP were transformed into in Arabidopsis thaliana.Some transgenic plants were obtained.The present results provide valuable data for subsequent investigation of inhibitory mechanisms of this glucanse and further use of the gene in genetic engineering of fungal resistance in plants.更多还原