中国农业科学 ›› 2017, Vol. 50 ›› Issue (21): 4071-4086.doi: 10.3864/j.issn.0578-1752.2017.21.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

D2型细胞质雄性不育小麦绒毡层细胞程序化死亡与活性氧代谢

刘子涵,石晓艺,闫鹏娇,段阳,耿兴侠,叶佳丽,李莎,杨雪桐,张高明,贾雨林,张玲丽,宋喜悦   

  1. 西北农林科技大学农学院,陕西杨凌 712100
  • 收稿日期:2017-04-10 出版日期:2017-11-01 发布日期:2017-11-01
  • 通讯作者: 宋喜悦,E-mail:songxiyue@126.com
  • 作者简介:刘子涵,E-mail:liuzihan643@163.com。
  • 基金资助:
    国家自然科学基金(31271792)

Tapetal Programmed Cell Death, Antioxidant Response and Oxidative Stress in Wheat Anthers Associated with D2-type Cytoplasmic Male-sterility

LIU ZiHan, SHI XiaoYi, YAN PengJiao, DUAN Yang, GENG XingXia, YE JiaLi, LI Sha, YANG XueTong, ZHANG GaoMing, JIA YuLin, ZHANG LingLi, SONG XiYue   

  1. College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi
  • Received:2017-04-10 Online:2017-11-01 Published:2017-11-01

摘要: 【目的】D2型细胞质是细胞质雄性不育小麦(cytoplasmic male sterility,CMS)的重要胞质来源,深入研究其败育机理对小麦杂种优势利用具有重要价值。【方法】以3种同核异质D2型细胞质雄性不育小麦Va87B1-706A、C687B1-706A、Ju87B1-706A及其相应保持系706B为试材,通过体式显微镜和DAPI染色分别在四分体时期、单核早期、单核晚期、二核期和三核期观察小麦花药的外形和小孢子发育的形态;利用扫描电镜和KI-I2染色分析三核期花药外皮层、内皮层、乌氏体和花粉粒的表型及败育特点;采用石蜡切片和DNA ladder的技术观察不同发育时期D2型不育系和保持系小麦花药绒毡层细胞形态变化特征以及绒毡层细胞程序化死亡(programmed cell death,PCD)的过程;测定花药发育过程中活性氧(reactive oxygen species,ROS)的含量,超氧化物歧化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)、过氧化氢酶(hydrogen peroxidase,CAT)、抗坏血酸过氧化物酶(ascorbate peroxidase,APX)和谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)的活性以及非酶物质还原型抗坏血酸(ascorbic acid,AsA)和谷胱甘肽(glutathione,GSH)的含量变化;同时利用RT-PCR技术对部分抗氧化酶相关基因的表达模式进行分析。【结果】与保持系706B比较,3种D2型细胞质雄性不育小麦在三核期均表现出外皮层表皮褶皱、乌氏体排列稀疏散乱以及小孢子皱缩、表皮粗糙和萌发孔闭合的不育特征,败育类型为典败和染败;自单核晚期开始3种不育小麦的小孢子发生紊乱;石蜡切片观察绒毡层细胞的变化,发现不育系均比保持系延迟一个时期在二核期发生解体,并于三核期完全降解;进一步检测细胞凋亡DNA小片段发现3种不育系花药绒毡层的DNA损伤均起始于单核晚期,绒毡层PCD比保持系延迟一个时期启动,并且优先细胞学表型检测到凋亡;生理指标测定和定量分析的结果说明绒毡层细胞的延迟凋亡和小孢子的结构异常与不育系花药内活性氧的过度积累、抗氧化酶活性的异常变化、非酶促抗氧化物质的严重下调以及部分抗氧化酶相关基因在两系花药中表达水平的严重差异密切相关。【结论】D2细胞质雄性不育小麦败育的关键时期是单核晚期,绒毡层PCD的延迟启动诱导了活性氧的过量积累,进一步破坏了抗氧化防御系统的平衡,进而造成绒毡层细胞形态异常,最终导致D2型细胞质雄性不育小麦的败育。

关键词: 小麦, D2型细胞质雄性不育, 绒毡层细胞程序化死亡, 活性氧代谢, 基因表达

Abstract: 【Objective】D2 type cytoplasm is the considerable source of cytoplasm of cytoplasmic male sterility (CMS). It is of great value to further study the cytological characteristics and mechanism of physiology and molecules in heterologous cytoplasmic male sterility (CMS) wheat for the heterosis utilization. 【Method】To elucidate the abortive cytological characteristics of D2-type cytoplasmic male-sterility, the phenotypes of anthers, microspores, tapetum were analyzed by scanning electron microscope, DAPI staining, and paraffin section, as well as detectingtapetal apoptotic DNA fragmentation by DNA ladder. To further clarify that the D2-type cytoplasmic male-sterility is associated with abnormal tapetal programmed cell death and reactive oxygen species metabolism, the physiological properties, including the contents of reactive oxygen species (ROS), activities of ROS-scavenging enzymes (superoxide dismutase (SOD), peroxidase, catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX)) and nonenzymatic antioxidants (ascorbic acid (AsA) and glutathione (GSH)), and the expression levels of antioxidant-associated genes from three D2-types of cytoplasmic male sterile lines (Va87B1-706A, C687B1-706A, Ju87B1-706A) and the maintainer 706B of wheat were compared.【Result】Compared with the maintainer line, at the trinucleate stage, the inner epidermal cells of D2-type cytoplasmic male-sterility accumulated less and more unconsolidated Ubisch bodies and the epidermis of microspores were extremely scabrous. The abortive pivotal period of pollen was initiated at the later uninucleate stage, which the delayed degradation of tapetum and abnormal structure of the microspore were related to the serious oxidative stress, disorganized activities of antioxidant enzymes, down-regulated contents of non-enzymatic antioxidants and significant difference of the expression level of some related enzyme genes in the three D2-types of cytoplasmic male sterile lines. Additionally, the expression level of SOD and APX genes was approximated to the activities of enzymes. However, there existed a distinct difference between the expression level of CAT gene and the activities of enzymes resulting from multiple-gene encoding CAT gene.【Conclusion】It was deduced that delay of start-up of the tapetal PCD (Programmed Cell Death) results from excessive ROS accumulation, and then destroyed the balance of the antioxidant defense system, and eventually led to the abortion of D2-types of cytoplasmic male sterile lines. Therefore, in order to further explore the abortive mechanism of cytoplasmic male sterility (CMS) wheat, this study provided an important theoretical basis and application significance.

Key words: wheat (Triticum aestivum L.), D2-types cytoplasmic male sterility, tapetal programmed cell death, ROS metabolism, gene expression