【作者】 管睿；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2010， 硕士

【摘要】 ‘红暗柳’甜橙(Citrus sinensis[L.]Osbeck)是一个特异性积累番茄红素的普通甜橙红肉突变体。‘红暗柳’与‘暗柳’的比较蛋白组学研究发现一个脂类相关质体蛋白(PAP)的表达伴随果实发育与色素累积显著上升,且在‘红暗柳’中的累积量高出暗柳两倍以上。质体相关蛋白是一大类古老而保守的蛋白质家族,参与脂蛋白结构如质体球蛋白和原纤维的合成,与质体分化和类胡萝卜素形成和储存密切相关。本研究旨在分离编码该蛋白的基因CSPAP并通过遗传转化的方法对其功能验证作初步的探索。主要工作和结果如下：1、在‘红暗柳’中克隆到CsRAP的cDNA全长为972 bp的片段,与已报道编码PAP蛋白的基因有很高的同源性；构建了CsPAP基因原核表达载体并在大肠杆菌中诱导了预测大小约为29 kD的特异蛋白的表达。2、构建了CsPAP基因的植物组成型表达载体,并以农杆菌介导的方法对国庆1号(Citrus unshiu)愈伤组织、山金柑(Fortunella hindsii)上胚轴以及番茄(Lycopersicon esculentum)子叶进行了遗传转化。通过筛选培养获得了国庆1号的抗性愈伤组织和番茄的再生抗性苗,经PCR检测为转基因阳性。3、Real-time PCR检测到一个抗性愈伤系中CsPAP的表达量比未转基因对照高出50倍以上。另外,ABA处理和逆境处理(NaCl.H202和脱水处理)能影响伏令夏橙愈伤组织中CsPAP基因的表达。更多还原

【Abstract】 ’Hong anliu’sweet orange (Citrus sinensis [L.] Osbeck) is a red-fleshed mutant dominantly accumulating lycopene. In the comparative proteomics study of’Hong anliu’ (MT, mutant) and’Anliu’(WT, wild type), a plastid lipid-associated protein (PAP) was detected to increase in parallel to the accumulation of pigments during the development of fruits and its expression in MT was over two-fold of that in WT. PAP is a large group of protein family involving in the formation of plastoglobules and fibrils, plastid differentiation and carotenoid sequestration. The research aimed at cloning the gene encoding CsPAP and primarily analyzing its function by genetic transformation.The isolated cDNA was 972 bp in length and had high identity rate with PAP genes reported in other species. The prokaryotic expression vector carrying cDNA fragment was constructed and transformed into E. coli to induce protein with a predicted molecular weight of 29 kD.The eukaryotic constitutive expression vector was also constructed to over-express CsPAP gene in callus (Citrus unshiu), epicotyls (Fortunella hindsii) and tomato cotyledon (Lycopersicon esculentum) by Agrobacterium-mediated transformation. Kanamycin-resistant citrus callus and regenerated tomato plants were obtained by selective culture and verified to be transgenic by PCR.Real-time PCR analysis revealed that CsPAP gene expressed over 50-fold in one line of transgenic citrus callus than in CK. Meanwhile, ABA treatment and certain stress conditions (NaCl, H2O2, and dehydration treatment) could bring out effects on the expression of CsPAP in Valencia callus (Citrus sinensis).更多还原