为建立适合红肉蜜柚发育过程中汁胞蛋白质组学的研究体系，以着色初始时期的红肉蜜柚汁胞为试材，摸索最适合的蛋白质双向电泳体系参数。结果表明，酚提取法比TCA-丙酮法更适合于红肉蜜柚汁胞总蛋白质的提取，提取得率高，双向电泳图谱清晰。采取18 cm、线性pH 3～10 和18 cm、线性pH 4～7 相结合的分析策略，可获得更为完整的蛋白质组信息，其第一向等电聚焦最佳参数（总伏小时数）分别为30 000 Vhs和43 000 Vhs。
In order to establish proteomics research system for analyzing ‘Hongroumiyou’ juice sacs proteins, using ‘Hongroumiyou’ juice sacs in initial color change stage as test material, exploring the most suitable 2-DE system parameters. The results indicated that compared with TCA-acetone extraction method, phenol extraction method was more appropriate for ‘Hongroumiyou’ juice sacs total protein yield. Using 18 cm, pH 4～7 linner immobiline combined with 18 cm, pH 3～10 linner immobiline could had clear and high resolution 2-DE patterns. Both of the best isoelectric focused parameter was 43 000 vhs and 30 000 vhs, respectively.
[1] 陆修闽,刘庆,徐娟,等. 红肉蜜柚果肉红色色素鉴定[J]. 亚热带植物科学, 2006,35(1): 39-41.
[2] 卢新坤,林旗华,陆修闽,等. 不同蜜柚果肉颜色类胡萝卜素成分及含量比较[J]. 福建农业学报, 2012,27(7): 723-727.
[3] 胡位荣,孙茹,温焱光,等. 响应曲面法优化红肉蜜柚番茄红素提取工艺的研究[J]. 广东农业科学, 2012(17): 86-89.
[4] 陈德欣. 红肉蜜柚β-胡萝卜素合成途径相关酶基因的克隆[D]. 福州: 福建农林大学硕士学位论文, 2007.
[5] Damerval C, De Vienne D, Zivy M, et al. Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat seedling proteins[J]. Electrophoresis, 1986,7(1): 52-54.
[6] 钟凤林. 琯溪蜜柚汁胞发育过程的差异蛋白质组学研究[D]. 福州: 福建农林大学博士学位论文, 2009.
[7] Saravanan R S, Rose J K. A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues[J]. Proteomics, 2004,4(9): 2522-2532.
[8] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem, 1976,72: 248-254.
[9] 李开拓. 荔枝果实成熟过程中的差异蛋白质组学研究[D]. 福州: 福建农林大学博士学位论文, 2011.
[10] Canovas F M, Dumas-Gaudot E, Recorbet G., et al. Plant proteome analysis[J]. Proteomics, 2004,4(2): 285-298.
[11] 陈晶瑜,郭宝峰,何付丽,等. 适合双向电泳的植物全蛋白提取方法比较[J]. 中国农学通报, 2010,26(23): 97-100.
[12] Nandakumar M P, Shen J, Raman B, et al. Solubilization of trichloroacetic acid (TCA) precipitated microbial proteins via NaOH for two-dimensional electrophoresis[J]. Journal of Proteome Research, 2003,2(1): 89-93.
[13] Carpentier S C, Witters E, Laukens K, et al. Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis[J]. Proteomics, 2005,5(10): 2497-2507.
[14] Valcu C M, Schlink K. Efficient extraction of proteins from woody plant samples for two-dimensional electrophoresis[J]. Proteomics, 2006,6(14): 4166-4175.
[15] 曾广娟,李春敏,张新忠,等. 苹果叶片蛋白质双向电泳样品制备方法的比较[J]. 中国农学通报, 2008,24(8): 105-109.
[16] 熊军波. 紫花苜蓿响应盐胁迫的蛋白质组研究[D]. 北京: 中国农业科学院博士学位论文, 2011.
[17] 李红兵,康振生. 适于小麦叶片蛋白质组分析的样品提取方法研究[J]. 西北植物学报, 2011,31(3): 632-638.
[18] 吕建珍. 沙田柚花粉壁蛋白和酸柚花粉壁蛋白的比较[D]. 桂林: 广西师范大学硕士学位论文, 2004.
[19] Pan Z, Liu Q, Yun Z, et al. Comparative proteomics of a lycopene-accumulating mutant reveals the important role of oxidative stress on carotenogenesis in sweet orange (Citrus sinensis [L.] osbeck)[J]. Proteomics, 2009,9(24): 5455-5470.
[20] Liu Q, Xu J, Liu Y, et al. A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis [L.] Osbeck)[J]. Journal of Experimental Botany, 2007,58(15-16): 4161-4171.
[21] Yun Z, Li W Y, Pan Z Y, et al. Comparative proteomics analysis of differentially accumulated proteins in juice sacs of ponkan (Citrus reticulata) fruit during postharvest cold storage[J]. Postharvest Biology and Technology, 2010,56(3): 189-201.
闽公网安备 35020602000789号