【作者】 高大玉；

【导师】 杨业华；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2005， 硕士

【摘要】 甘露糖基转移酶（mannosyl transferase,mant）基因是从拟南芥克隆而来,普遍存在于高等真核生物的基因组中,其编码产物的基本功能是将甘露糖从GDP-甘露糖转移到长醇一磷酸酯上,形成长醇磷酸甘露糖。在内质网泡内,长醇磷酸甘露糖则是N-糖基化途径、糖磷脂酰肌醇膜固定途径以及蛋白质的O-甘露糖基化的甘露糖基的供体。甘露糖也是聚唾液酸和植物外源凝集素（lectin）的成分,而这两种甘露糖蛋白又是植物的细胞壁成份之一,他们都与植物的抗虫和抗病性有关。因此甘露糖代谢的异常,也会导致植物对病原微生物及害虫抗性特征的改变以及植物本身生长发育的异常。本研究利用mant基因的编码序列与花药特异启动子、含增强子四联体的花药特异启动子构建成融合基因和该基因的反义RNA基因,并通过农杆菌介导法将这些融合基因导入棉花基因组中,使其超表达、花药特异表达或抑制棉花同源基因的表达,以期创造出新的抗病、抗虫和转基因雄性不育系。 本研究取得的实验结果如下: 1.通过农杆菌介导的遗传转化方法将mant基因的上述三种转化结构成功导入棉花基因组中,共获得转化植株66株。 2.分别以NPTⅡ和mant基因序列设计引物,对获得的当代转基因植株进行PCR检测。NPTⅡ设计的特异引物筛选阳性率为59.1%,mant设计的特异引物筛选阳性率为50%,两对引物筛选的共阳性率为36.4%。 3.对转基因PCR阳性植株进行Southern杂交检测,其中NPTⅡ特异探针的杂交结果表明,无论棉花基因组DNA以kpnI或EcoRI酶切,T₀代不同植株间探针杂交阳性片段大小各异,多态性表明外源基因在棉花基因组中整合位点不同,转基因植株的嵌合程度较低。Mant特异探针杂交结果表明,棉花基因组DNA以EcoRI酶切,都可见预期大小的内部片段,并且初步证明棉花基因组存在mant同源序列。这些结果均表明外源基因确已经整合到棉花基因组中。 4.对转基因当代植株进行生育特性考查,在育性方面并未表现出与对照有明显差别,抗虫、抗病特性以及雄性不育性的转基因植株有待从T1代转基因群体中进行筛选、鉴定。更多还原

【Abstract】 Mannosyl transferase（mant） is cloned from Arabidopsis Arabidopsis thianala. It is believed that the gene encoding Mannosyl transferase（mant） universally exists in the genomes of all eukaryotic and prokaryotic organisms. Its coding product Mannosyl transferase transfers mannose from GDT-mannose to dolichol monophosphate to form dolichol phosphate mannose （dol-p-man）, which serves as a donor of mannosyl residues on the lumenal side of the endoplasmic reticulum leading to N-glycosylation, glycosyl phosphatidylinositol membrane anchoring, and O-mannosylation of proteins. Mannose also is the basic component of saliva acid and plant lectin. These two kinds of glycoproteins are parts of plant cell wall components and highly related to disease and insect resistances of plants. Therefore, abnormalities of mannose metabolisms certainly result in the changes of plant resistibility to virulent microorganisms and pests, and form barriers to plant normal developments. In this study, the coding sequence of the mant gene was applied to fuse with anther-specific promoter, tetravalent enhancer plus anther-specific promoter to build the constructs of fused genes and antisense RNA gene, and with which to transform apex segments of upland cotton via Agrobacterium mediated transformation, and to make them over expression, anther specific expression, or suppress the expression of the homologues counterparts in the cotton genome in the purpose of creating of new disease- and insect- resistant cotton varieties and transgenic male sterile lines.The main results in this study were as follows:1. The above-mentioned three mant constructs were successfully transferred to the cotton genome by Agrobacterium mediated transformation. Total 66 T₀ transgenic plants were obtained.2. The primers were designed according to the sequences of NPTⅡand mant gene and applied to PCR analyses of To transgenic plants. Percentage of the plants with positive signals in nptⅡ primer reaction is 59.1%, that of the plants in mant primer reaction is 50%, and the percentage of the plants showing both positive signals of nptll and mant primer reactions is 36.4%.3. The T₀ transgenic plants displayed positive signals in PCR reaction were further analyzed in Southern blotting. The results showed that whatever the genomic DNAs digested with restriction enzymes of KpnI or EcoRI both had the polymorphisms of restriction fragments while probed with the PCR product of nptll gene. But the size of the fragments only varied 9kb-25kb among the plants, indicating that the foreign geneshad integrated into the different sites of the host genome, and that the To plants originated from different transformed cells and had a lower degree of chimera.The hybridization results taking the PCR fragment of mant gene as probe showed that digestions of EcoRl presented internal fragments with anticipated sizes.4. Field characterization of TO transgenic plants did not show any significant difference from the check test plants in phenotypes, fertility and pollen grain development. It is possible that the transgenic mutants with desirable traits, such as insect- and disease-resistances and male sterility, could be identified and isolated from the Tl segregating populations.更多还原