【作者】 刘峰；

【导师】 赖钟雄；

【作者基本信息】 福建农林大学 ， 细胞生物学， 2012， 硕士

【摘要】 本研究以葡萄（Vitis vinifera）为试验材料，利用组织培养的技术针对福建省的葡萄种质资源进行收集培养得到葡萄试管苗，并进行离体保存，探索最佳的离体保存条件。本论文主要进行了以下几方面的研究：①葡萄试管苗离体再生体系的建立，②葡萄试管苗离体保存的研究，③葡萄试管苗离体保存过程中蛋白质组学的研究。具体如下：1葡萄试管苗离体再生体系的建立试验中以葡萄单芽茎段为外植体材料，最佳的取材时间是春季4月份新枝刚抽出时。以美人指单芽茎段为材料对比不同的初代培养基组合，结果表明以不添加细胞分裂素，无机盐浓度较低的GS+0.2mg/L IAA的培养基为好。同一培养基对不同葡萄进行初代培养，结果表明不同基因型对芽的诱导率影响起着重要作用。不同基本培养基，不同6-BA和IAA浓度的正交设计增殖培养基的筛选中以MS（1/2N）+1mg/L6-BA+0.1mg/L IAA最佳，增殖系数达到了6.8。2葡萄试管苗离体保存试验以美人指试管苗为材料研究不同浓度多效唑和不同浓度甘露醇对试管苗离体保存影响的单因素试验。结果表明多效唑和甘露醇都能够有效地限制试管苗的生长，最佳的多效唑浓度为2mg/L，最佳的甘露醇浓度是15g/L。以GS+2mg/L多效唑为培养基接种10个不同葡萄品种试管苗，结果表明此浓度多效唑对多数基因型葡萄都有很好的限制生长作用。另外，以白砂糖、甘露醇、多效唑为因子设计了三因素三水平的正交试验，结果表明最佳的组合是：GS+30g/L白砂糖+2.5mg/L多效唑+14g/L甘露醇。3葡萄离体保存过程中蛋白质组学的研究①葡萄试管苗离体保存过程中蛋白质的双向电泳将同一株系的美人指试管苗接种在试验组：GS+2mg/L多效唑+30g/L蔗糖+6g/L琼脂，对照组：GS+30g/L蔗糖+6g/L琼脂；分别在10天、20天、30天、40天时取材，蛋白提取、定量后进行双向电泳。对得到的电泳图谱利用Image master2D Platinum6.0软件进行分析，试验组得到的蛋白点个数分别为：356、368、286、174，对照组得到的蛋白点个数分别为：261、295、183、154。10天时，对照组中特异表达的蛋白质比试验组多80个，而在后面阶段试验组特异表达的蛋白质都要比对照组中多。从分子量的分布情况来看大部分蛋白的分子量为30-45KDa之间。②葡萄试管苗离体保存过程中相关蛋白的质谱鉴定及功能分析试验选取葡萄试管苗离体保存过程差异表达的49个蛋白点进行了质谱鉴定。最终，成功鉴定46个，占93.9%。葡萄基因组测序在2007年已经完成，因此质谱鉴定成功蛋白中有很多是预测蛋白或假定蛋白，占74%。蛋白的功能分类表明差异蛋白主要集中在了与光合作用有关的细胞组分中。从鉴定蛋白所参与的生物过程来看主要集中在氧化还原，CO₂的固定，碳水化合物代谢过程，防御反应，应对生物刺激等方面。从分子功能角度来看鉴定成功蛋白主要的功能集中在催化活性，二磷酸核酮酶活性，氧化酶活性，核酸结合活性等。在已知蛋白中多数与抗氧化作用和光合作用有关。表明多效唑能够增强葡萄试管苗的抗性。更多还原

【Abstract】 In this experiment the grape（Vitis vinifera）was used as the experimental material. somegrape germplasm resources were collected in Fujian Province to conduct the following researches①establishment of the in vitro regeneration system in grape；②the in vitro conservation ofgrape germplasm；③proteomics researchduring grape in vitro conservation.1. Establishment of in vitro regeneration system in grape.The stem segments with a single node were used as explants in Vitis vinifera cv. ManicureFinger. The best season for sanpling was April. The primary culture showed that the best mediumwas GS Medium supplemented with0.2mg/L IAA. The genotype affected the induction rates inthe primary culture. The best medium for proliferation was MS(1/2N) Mediun with1.0mg/L6-BAand0.1mg/L IAA, with the proliferation rate as high as6.8on the30thday.2. In vitro conservation in grape.The effects of different concentrations of paclobutrazol and mannitol on the in vitroconservation were compared in in Vitis vinifera cv. Manicure Finger. The result showed that thebest conservation effect occurred on the medium GS supplemented with0.2mg/L PP333or15g/LMannitol. The comparisons of in vitro conservation of10grape cultivars cultered on the GSMedium supplemented with2mg/L PP333indicated that there were similar effects among them.The orthogonal experiment of the different concentrations and combinations of mannitol and PP333and sugar showed that GS Medium supplemented with30g/L suger,2mg/L PP333and14g/Lmannitol was optimum for grape in vitro conservation.3. Proteomics research during in vitro conservation in grape.①2-D electrophoresis of the plantlet proteins during in vitro conservation in grape.Two-dimensional gel electrophoresis coupled to mass spectrometry analysis was used toexamine for the first time during the in vitro conservation in grape. The same line of in vitroManicure Finger planlets was used in the test. Tube plantlets were cultured on GS Medium with 2mg/L PP333or not. Control and PP333-treated plantlets were sampled after10,20,30and40dafter transfer to the new medium. Examination of2-D maps derived from PP333-treatmentreavealed the presence of many spots displaying a differential expression pattern. The PP333-treatment group detected spots number was356,368,286and174. However, the control group was261,295,183,154, respectively. The MWs of most proteins were30-45kDa.②Identification of proteins by mass spectrometry and analysis of their functionsduring in vitro conservation in grape.49protein spots were selected for mass specteometry analysis.46protein spots weresuccessfully identified. Among the responsive proteins, some photosynthsis-related proteinsincluding several fragments of the enzyme Rubisco were identified. Several enzymatic antioxidantand pathogenesis-related proteins were also observed. The proteins which were successfullyidentified were involved in many biology processes, which mainly concentrated on oxidation/reduction, carbohydrate metabolic process, defense response. Meanwhile, from the point view ofthe molecular function, the proteins were concentrated on catalytic activity, oxidoredactase activity,magnesium ion binding, nucleotide binding. Among the known proteins, most were related to anti-oxidization and photosynthsis, which suggested that adding paclobutrazol improve that resistanceof grape plantlets.更多还原