中国农业科学 ›› 2016, Vol. 49 ›› Issue (8): 1530-1539.doi: 10.3864/j.issn.0578-1752.2016.08.010

• 园艺 • 上一篇    下一篇

着色与非着色苹果品种中磷酸化蛋白质的鉴定与分析

苑克俊,程来亮,牛庆霖,王江勇   

  1. 山东省果树研究所,山东泰安 271000
  • 收稿日期:2015-12-23 出版日期:2016-04-16 发布日期:2016-04-16
  • 通讯作者: 苑克俊,Tel:0538-8238791;E-mail:yuankj@vip.sina.com
  • 作者简介:苑克俊,Tel:0538-8238791;E-mail:yuankj@vip.sina.com
  • 基金资助:
    植物分子遗传国家重点实验室开放课题、山东省泰山学者建设项目

Identification and Analysis of Phosphoproteins in Red and Non-Red Apple Cultivars

YUAN Ke-jun, CHENG Lai-liang, NIU Qing-lin, WANG Jiang-yong   

  1. Shandong Institute of Pomology, Tai’an 271000, Shandong
  • Received:2015-12-23 Online:2016-04-16 Published:2016-04-16

摘要: 【目的】苹果着色是一个重要的质量性状,构建苹果磷酸化蛋白质组并进行鉴定与分析,了解在苹果着色过程中的蛋白质磷酸化,为进一步阐明苹果着色机理提供参考。【方法】以着色‘嘎拉’和非着色‘金冠’两个品种苹果果皮为试材提取总蛋白,用TiO2富集磷酸化肽段,对富集到的磷酸化肽段进行高效液相色谱分离,利用质谱鉴定磷酸化肽段,采用Mascot2.2和Proteome Discoverer1.4(thermo)软件进行蛋白质查库鉴定,使用的数据库为Uniprot_Maloideae.fasta。对鉴定到的磷酸化肽段和蛋白质进行分析,找出着色与非着色苹果品种中有差异的磷酸化蛋白质及其修饰位点。【结果】在着色苹果品种中鉴定到1 323个蛋白质、3 339个肽段和225个磷酸化肽段,发现磷酸化蛋白质约200—225种;在非着色苹果品种中鉴定到569个蛋白质、1 152个肽段和128个磷酸化肽段,发现磷酸化蛋白质约117—128种。比较分析表明,43个磷酸化肽段仅在非着色苹果品种中发现,139个磷酸化肽段仅在着色苹果品种中发现,在着色苹果品种中有更多的蛋白质发生了磷酸化,其中质膜H+-ATPase在两个苏氨酸位点发生磷酸化,过敏原蛋白和两个蛋白激酶在丝氨酸位点发生磷酸化,3个转录因子、1个钾转运蛋白和1个受体激酶都发生磷酸化。在两个苹果品种鉴定出的蛋白质中,发生蛋白质磷酸化的位点主要是丝氨酸,其次是苏氨酸,在酪氨酸位点发生蛋白质磷酸化很少。蛋白质WD40和花青苷合成途径中的相关酶蛋白C4H、4CL、CHS、CHI、F3′H、FLS、LDOX和UFGT在着色过程中未发生磷酸化。在着色和非着色苹果品种中都存在MYBR转录因子,并且都发生了蛋白质磷酸化。【结论】在着色苹果品种中产生了一些特有的磷酸化蛋白质,其中包括已知与着色相关的质膜H+-ATPase,苹果着色可能受到蛋白质磷酸化的影响。

关键词: 苹果, 磷酸化, 蛋白质组, 蛋白激酶, 着色

Abstract: 【Objective】Apple skin color is an important fruit quality factor. In order to make clear whether there is phosphorylation during apple fruit pigmentation, large scale identification of phosphoproteins and phosphopeptides was conducted. 【Method】The fruit peels of red skinned apple cultivar ‘Gala’ and non-red skinned apple cultivar ‘Golden Delicious’ were used to extract protein. Fruit protein was digested with trypsin, the resulting peptides were subjected to a step of TiO2 beads enrichment to obtain phosphopeptides, and a step of HPLC separation.Q-Exactive mass spectrometer was used to identify phosphopeptides. The software Mascot 2.2 and Proteome Discoverer1.4 (thermo) were used to identify proteins in the database Uniprot_Maloideae.fasta. Then data analysis was performed to provide information of identified phosphorylation sites in red and non-red apple cultivars.【Result】 1 323 proteins, 3 339 peptides, 225 phosphopeptides were identified and about 200-225 phosphoproteins were found in the red apple cultivars, 569 proteins, 1 152 peptides, 128 phosphopeptides were identified and about 117-128 phosphoproteins were found in the non-red apple cultivars.Comparative analysis indicated that 43 phosphopeptides were only found in non-red apple cultivars, 139 phosphopeptides were only discovered in red apple cultivars, which proved that more phosphoproteins produced in red apple cultivars, including plasma membrane H+-ATPase with two phosphorylation sites in serine, a major allergen and two protein kinases with phosphorylation site in threonine, three transcription factors, a potassium transporter and a putative protein receptor kinase. Among the identified proteins in the red and non-red apple cultivars, the phosphorylation sites were mainly found in threonine, some in serine, seldom in tyrosine. There was no phosphorylation site in the C4H, 4CL, CHS, CHI, F3′H, FLS, LDOX and UFGT proteins of anthocyanin synthesis pathway and protein WD40, the MYBR transcription factor with phosphorylation site was found in both red and non-red apple cultivars. 【Conclusion】 Some phosphoproteins only produced in red apple cultivars, including plasma membrane H+-ATPase related to apple pigmentation. Apple fruit pigmentation is possibly influenced by protein phosphorylation.

Key words: apple, phosphorylation, proteome, protein kinase, pigmentation