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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2015, Vol. 41 Issue (1): 44-55    DOI: 10.3785/j.issn.1008-9209.2014.04.242
生物科学与技术     
辣椒胞质不育系与保持系花药的细胞学和蛋白质组学差异分析
吕晓菡1, 方献平2, 柴伟国1*, 马俊平3, 周毅飞1
1.杭州市农业科学研究院蔬菜研究所, 杭州310024;2.杭州市农业科学研究院生物技术研究所, 杭州310024;3.浙江大学农业与生物技术学院,杭州 310058
Comparative analysis of cytology and proteomics in anthers between cytoplasmic male sterile line and its maintainer line of peppers
Lü Xiaohan1, Fang Xianping2, Chai Weiguo1*, Ma Junping3, Zhou Yifei1
(1. Institute of  Vegetables, Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, China; 2. Institute of Biology, Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, China; 3. College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China)
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摘要： 为了解辣椒胞质雄性不育系与保持系花药的细胞学和蛋白质组学差异,以辣椒胞质不育系和保持系花蕾发育的6个不同时期的花药为材料,以石蜡切片于Leica DM1000显微镜下观察其发育过程的差异。然后利用SDS-PAGE进行花药蛋白分离,经LC-MS/MS质谱技术和蛋白质组学分析对差异蛋白质条带进行鉴定。结果显示:花粉败育发生在四分体形成以后,与绒毡层细胞的过度液泡化及四分体周围的胼胝质不能解体有关。另外,与辣椒保持系相比,在不育系花药蛋白中有4条差异蛋白质条带（条带1～4）表达量下调,质谱分析后共鉴定出蛋白质68个,其中不重复蛋白质64个。对质谱数据利用UniProt数据库进行搜索鉴定,鉴定出的蛋白质按分子功能归类,涉及催化作用的蛋白质最多有24个;按细胞组分归类,涉及细胞内膜组织的最多有20个。按生物学过程归类,参与代谢过程的最多有24个。这说明在四分体形成以后,胞质雄性不育系花药的物质和能量代谢出现异常,导致绒毡层异常膨大,四分体不能获得物质和能量从而败育。另外,这也充分说明本研究通过蛋白质组学分析鉴定出的差异蛋白质可初步解释胞质雄性不育系与保持系在细胞学上的差异表现。
Abstract: Cytoplasmic male sterility （CMS） lines are the excellent materials for studying the cytoplasmic and nuclear cytoplasmic interaction and have important application and theoretical value for CMS research. Three-line hybrid seed production could not only reduce the trouble of manual emasculation and seed costs, but also protect the purity of hybrid seeds. Therefore, the production of hybrid seed using CMS lines has a great potential and market demand in the pepper production.With the continuous development of science and technology, proteomics has become an important tool to decipher the biological gene function and contribute to the plant breeding. Proteome analyses helped to explore the important plant genes, thus speeding up the process of plant breeding and genetics research. However, the current researchs on male sterile pepper were mostly concentrated in the physiological and biochemical indexes and the male sterile gene. The relevant report on CMS pepper protein was relatively less, and the research of sterile anther protein was even more rarely reported. Therefore, the aim of this study was to preliminarily identify the differences of cytology and proteomics involved in the peppers anther of CMS line and its maintainer line. The anthers at six different stages were used in the experiment. At first, the paraffin sections of anthers were observed and used to study the differences between CMS line and its maintainer line from the apparent characteristics, and then SDS-PAGE was used for anther protein separation and identification. Differential protein bands separated by SDS-PAGE, and the proteome profiles of the differential protein bands were further analyzed and identified by LC-MS/MS and proteomics analysis. The results showed that pollen abortion occurred after metaphase. The main reason was that the callose surrounding the tetrad didnt disintegrate normally and the tapetum cells were excessive vacuolation. One-dimensional SDS-PAGE analysis revealed that, compared with its maintainer line, there were four fewer protein bands in CMS line. A total of 64 non-redundant proteins were identified by LC-MS/MS, including 12 unique proteins from band 1, 9 unique proteins from band 2, 21 unique proteins from band 3, 18 unique proteins from band 4, and 2 proteins from both band 1 and band 3, 1 protein from both band 2 and band 3, 1 protein from both band 3 and band 4. Among them, 24 proteins were involved in catalysis according to molecular function; 20 proteins were involved in cell endometrial tissue according to cellular components; and 24 proteins were involved in the metabolic process according to biological processes. In conclusion, pepper CMS is mostly caused by pollen abortion. Pollen sterility is directly or indirectly related to abnormal tapetum and the lack of callose disintegration. After the differential proteome analysis between pepper CMS line and its maintainer line, we find that after metaphase, due to the decreased expression involving substance and energy metabolism in a part of the protein in the anther of male sterile line, the tapetum cells develop abnormally and the four separate microspores in development can not obtain matter and energy. Then pollen might be abortion. Therefore, in this study, the differential proteins identified by proteomics analysis can preliminarily explain the differences in cytology.
出版日期: 2015-01-20
CLC:  Q 246  
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柴伟国
马俊平
方献平
周毅飞
吕晓菡

引用本文:

吕晓菡, 方献平, 柴伟国, 马俊平, 周毅飞. 辣椒胞质不育系与保持系花药的细胞学和蛋白质组学差异分析[J]. 浙江大学学报(农业与生命科学版), 2015, 41(1): 44-55.

Lü Xiaohan, Fang Xianping, Chai Weiguo, Ma Junping, Zhou Yifei . Comparative analysis of cytology and proteomics in anthers between cytoplasmic male sterile line and its maintainer line of peppers. Journal of Zhejiang University (Agriculture and Life Sciences), 2015, 41(1): 44-55.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2014.04.242    或    http://www.zjujournals.com/agr/CN/Y2015/V41/I1/44

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