【作者】 王生毅；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 本研究在前期构建的欧洲葡萄‘无核白’胚珠cDNA文库的基础上,通过噬菌体侵染宿主菌XL1-Blue筛选阳性克隆后,以大肠杆菌BM25.8宿主菌进行培养,实现阳性克隆转换,对阳性克隆进行测序获得EST序列,并对特异序列进行鉴定,取得的主要结果如下:1.从欧洲葡萄‘无核白’胚珠cDNA文库中随机挑选了4800个克隆进行5’端测序,经剔除载体、污染序列和小于100 bp的序列后,共获得了3908条EST,这些序列登陆了NCBI GenBank数据库,这些序列及登陆号列入附录。2.对3908条EST聚类分析获得了3046个独立序列。这些独立序列对比拟南芥功能分类标准,共有2056个独立序列分为21类。其中与新陈代谢有关的有435个独立序列,能量有关的22个独立序列,储藏蛋白有关的6个,细胞周期和DNA加工有关的82个,翻译有关的110个,蛋白质合成有关的138个,蛋白质命运(折叠、修复、靶向运输)有关的183个,结合或辅助功能蛋白(结构的或催化的)有关的279个,新陈代谢调控和蛋白调控有关的11个,细胞运输、运输功能和运输路径有关的101个,细胞交谈/信号转导机制有关的10个,细胞自救、防御和抗病有关的39个,与环境间的相互作用有关的21个,系统与环境间的相互作用有关的1个,细胞命运有关的6个,系统发育有关的15个,细胞组分的生物进化有关的3个,器官分化有关的1个,亚细胞定位有关的158个,不明确的分类有关的126个,未获得分类的309个。3.进一步对获得的3046个独立序列与NCBI GenBank nr蛋白质数据库进行同源比对(E-value≤1e-5),共获得了69个与种子或胚发育相关的蛋白基因的独立序列,其中涉及与未受精胚囊蛋白基因(Unfertilized embryo sac)有关的独立序列3个,体细胞胚胎发生相关蛋白基因(Somatic embryogenesis related protein genes)有关的独立序列15个,胚发育缺陷蛋白基因(Embryo Defective genes)有关的独立序列17个,胚囊发育停止蛋白基因( Embryo sac development arrest genes)有关的独立序列7个,细胞程序性死亡(death-related protein gene)相关基因有关的独立序列6个,钙结合蛋白(Calcium-binding protein gene)相关基因有关的独立序列21个。4.从获得与细胞程序性死亡和钙结合蛋白相关的27个独立序列中分离了一个序列,该序列在无核与有核葡萄胚珠发育过程中表达差异显著。通过Race技术扩增,获得该序列全长为907 bp(登陆GenBank accession no. HQ214155),拥有2个EF-hand钙结合蛋白模体,简称之为VvCBP1基因,该基因序列与番茄同源基因的同源性为100%。利用VIGS技术在番茄中沉默VvCBP1的同源基因以间接验证VvCBP1的功能。研究结果表明,瞬时沉默番茄果实中种子数量明显减少。这个结果暗示,VvCBP1对番茄种子发育进程中有促使幼胚败育作用。更多还原

【Abstract】 This study was conducted on the basis of the cDNA library previously constructed from ovules of Vitis vinifera cv. Thompson Seedless. Positive clones were screened after bacteriophage infection of XL1-Blue and further culture with BM25.8 as host bacteria. The obtained positive clones were selected for sequencing and the isolated specific sequences were further identified. The main results are described as follows.1. 4800 clones from the cDNA library of ovules of V. vinifera cv.Thompson Seedless were randomly selected for 5’end sequencing. After removing vector sequences, polluted sequences and low quality sequences less than 100 bases, a total of 3908 high quality ESTs were obtained and have been deposited in NCBI GenBank database with accession number(Appendix).2. The 3908 ESTs were assembled into 3046 unique sequences. These unique sequences were aligned with the functional classification system of Arabidiopsis thaliana, and 2056 unique ESTs could be classified into 21 categories. Among them, the number of classified ESTs associated with metabolism, Energy, Storage protein, Cell cycle and DNA Processing, transcription, Protein synthesis, Protein fate(folding, modification, destination), Protein with binding function or cofactor requirement, Regulation of metabolism and protein function, Cellular transport, Transport facilitation and transport routes, Cellular communication/ signal transduction mechanism, Interaction with the environment, Systemic interaction with the environment, Cell fate, Development, Biogenesis of cellular components, Organ differentiation and Subcellular localization, were 435, 22, 6, 82,110, 138,183, 279, 11, 101, 10, 21, 1, 6,15,3, 1 and 158,respectively. 126 ESTs could not fell into the well-defined classification and 309 ESTs could not be were unclassified.3. The 3046 ESTs were further analyzed and 69 unique ESTs involving in seed or embryo development were obtained using the BLASTX against the NCBI non-redundant protein database (E-value≤1e-5). Among them, the number of ESTs involved in unfertilized embryo sac, Somatic embryogenesis related protein, Embryo Defective, Embryo sac development arrest genes, Cell death-related protein, and Calcium-binding protein, were 3, 15, 17, 7, Cell death-related protein 6, and 21, respectively. 4. A novel gene was found from 27 unigenes involving in Cell death-related protein genes and Calcium-binding protein, and its expression pattern in embryo is significantly different between seedless and seeded grapevine species. Based on the obtained partial EST sequence, a RACE-PCR method was performed to isolate the full-length gene. The result indicated that the gene is 907 bp in length, and possesses two EF-hand Calcium-binding motifs, and designated VvCBP1, which shares high similarities with that of the tomato. Using tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) technology, we silenced the tomato homolog of VvCBP1 in tomato fruits, which led to the number of injected tomato seed significantly decrease. These results suggest that VvCBP1 may be associated with fruit seedlessness of tomato.更多还原