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作物学报 ›› 2011, Vol. 37 ›› Issue (07): 1134-1143.doi: 10.3724/SP.J.1006.2011.01134

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦生理型和遗传型雄性不育系及其保持系小花完整叶绿体蛋白质组分比较研究

李莉,王书平**,张改生*,王亮明,宋瑜龙,张龙雨,牛娜,马守才   

  1. 西北农林科技大学陕西省作物杂种优势研究与利用重点实验室 / 小麦育种教育部工程研究中心，陕西杨凌 712100?
  • 收稿日期:2010-12-23 修回日期:2011-03-27 出版日期:2011-07-12 网络出版日期:2011-05-11
  • 通讯作者: 张改生, E-mail: zhanggsh@public.xa.sn.cn, Tel: 029-87092152
  • 基金资助:

    本研究由国家高技术研究发展计划(863计划)重大专项(2009AA101102), 国家自然科学基金项目(31071477), 高等学校博士学科点专项科研基金(20090204110024), 陕西省“13115”科技创新工程重大科技专项(2010ZDKG-68)和西北农林科技大学拔尖人才支持计划项目资助。

Comparison of Floret Intact Chloroplast Proteome in Male Sterile Line induced by CHA-SQ-1, Cytoplasmic-Nuclear Sterile and Its Normal Fertile Lines in Wheat

LI Li,WANG Shu-Ping**,ZHANG Gai-Sheng*,WANG Liang-Ming,SONG Yu-Long,ZHANG Long-Yu,NIU Na,MA Shou-Cai   

  1. Key Laboratory of Crop Heterosis of Shaanxi Province, Northwest A&F University / Wheat Breeding Engineering Research Center, Ministry of Education, Yangling 712100, China
  • Received:2010-12-23 Revised:2011-03-27 Published:2011-07-12 Published online:2011-05-11
  • Contact: 张改生, E-mail: zhanggsh@public.xa.sn.cn, Tel: 029-87092152

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摘要： 采用固相pH梯度SDS聚丙烯酰胺双向凝胶电泳技术，以小麦遗传型雄性不育系ms(S)-1376、杀雄剂SQ-1诱导的生理型雄性不育系ms(A)-1376，以及对应的保持系(A)-1376 (杀雄剂诱导前的正常可育系)所构建的等基因和等生理差异系为试材，比较分析了2种分离纯化完整叶绿体的方法。在确立了一套适用的技术方法的基础上，研究了三者在花粉小孢子萌发单核早期小花完整叶绿体蛋白之间的差异。采用30%、45%和60%的不连续蔗糖密度梯度离心的方法能够获得完整且纯度较高的叶绿体，经TCA-丙酮提取蛋白质后，PDQuest软件分析，在分子量14.4~66.2 kD，等电点4~7的线性范围内可分辨出2-DE图谱上239个较为清晰的蛋白质点，对其中的6个差异表达蛋白质点进行MALDI-TOF肽质量指纹图谱分析，从生物信息学数据库检索鉴定出6个差异蛋白质点分别是酰基辅酶A脱氢酶结构域蛋白、钙调结合蛋白磷酸酶、多催化功能肽酶、热休克蛋白60、光受体蛋白2及1个未知功能的表达蛋白质。这些蛋白质在供试小麦叶绿体物质能量代谢、叶绿体防卫抵御机制、叶绿体细胞内信号转导及植物极性生长等生理应答反应中起调控作用，它们能在本研究供试两不育系和对应可育系中差异表达，可能与雄性不育相关。

关键词: 小麦, 遗传型和生理型雄性不育, 完整叶绿体蛋白质组, 差异表达, 2D-PAGE, MALDI-TOF-MS

Abstract: Weextracted floret chloroplast proteins from the genetic male sterile line ms(S)-1376, the physiological male sterile line ms(A)-1376 induced by chemical hybridizing agent SQ-1, and their counterpart maintainer line (A)-1376 (normal fertility). These proteins were separated via 2-D electrophoresis with immobilized pH 4–7 gradients as the first dimension and SDS-PAGE as the second one. We established a set of technique available for intact chloroplast isolation and 2-DE of proteins in wheat floret and compared the effect of intact chloroplast purification using two methods. Based on the 2-DE technique, we explored the difference of the floret chloroplast proteins among the three materials tested in their early uninucleate anther stage. The result showed that we obtained high purity of intact chloroplast using discontinuous sucrose density gradient centrifugation with three-step gradient densities of 30%, 45%, and 60%. Proteins were extracted from chloroplast by TCA-acetone and analyzed by PDQuest software, we got as much as 239 protein spots within pH 4–7 and with molecular weight of 14.4–66.2 kD on each 2-DE gel. We analyzed six differential expressed proteins by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and bioinformatical database, and found they were acyl-CoA dehydrogenase domain protein, calmodulin-binding protein phosphatase, multiple catalytic peptidase, heat-shock protein 60, light receptor protein 2 and a protein of unknown function. These proteins are involved in series of physiological reactions such as metabolism of energy substances, chloroplast defendance, chloroplasts signal transduction and plant growth, the differential expression of those proteins among the three materials tested is likely related to the male sterility in wheat.

Key words: Wheat, Genetic and physiological male sterilities, Intact chloroplast proteins, Differential expression, 2-dimensional electrophoresis, MALDI-TOF-MS

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