小麦生理型雄性不育系微丝骨架和胼胝质的变化与其相关基因的表达分析

doi:10.3864/j.issn.0578-1752.2015.14.001

中国农业科学 ›› 2015, Vol. 48 ›› Issue (14): 2687-2696.doi: 10.3864/j.issn.0578-1752.2015.14.001

• 作物遗传育种·种质资源·分子遗传学 • 下一篇

小麦生理型雄性不育系微丝骨架和胼胝质的变化与其相关基因的表达分析

张姣¹，朱启迪^1,2，巨岚¹，张改生¹，于永昂¹，牛娜¹，王军卫¹，马守才¹

¹西北农林科技大学农学院/国家杨凌农业生物技术育种中心/国家小麦改良中心杨凌分中心/小麦育种教育部工程研究中心/陕西省作物杂种优势研究与利用重点实验室，陕西杨凌 712100
²河南科技学院生命科技学院，河南新乡 453003

收稿日期:2015-01-12 出版日期:2015-07-16 发布日期:2015-07-16
通讯作者: 张改生，E-mail：zhanggsh58@aliyun.com
作者简介:张姣，E-mail：zhangjiaoycm@163.com。朱启迪，E-mail：zzhhqqdd@163.com。张姣和朱启迪为同等贡献作者。
基金资助:
国家高技术研究发展计划重大专项（2011AA10A106）、国家支撑计划（2015BAD27B01）、国家自然科学基金（31171611、31371697）、陕西省科技统筹创新工程计划课题（2014KTZB02-01-02）

Organization Changes of Actin Cytoskeleton and Callose with the Expression Analysis of Related Genes in Physiological Male Sterile Wheat

ZHANG Jiao¹, ZHU Qi-di^1,2, JU Lan¹, ZHANG Gai-sheng¹, YU Yong-ang¹, NIU Na¹, WANG Jun-wei¹, MA Shou-cai¹

¹College of Agronomy, Northwest A&F University /National Yangling Agricultural Biotechnology & Breeding Center /Yangling Branch of State Wheat Improvement Centre /Wheat Breeding Engineering Research Center, Ministry of Education /Key Laboratory of Crop Heterosis of Shaanxi Province, Yangling 712100, Shaanxi
²College of Life Science and Technology, Henan College of Science and Technology, Xinxiang 453003, Henan

Received:2015-01-12 Online:2015-07-16 Published:2015-07-16

摘要/Abstract

摘要： 【目的】研究生理型雄性不育小麦花粉细胞内微丝和胼胝质的结构及其相关基因的表达，并揭示其与生理型雄性不育的关系，为进一步研究化学杂交剂SQ-1诱导小麦生理型雄性不育的机理提供一定的理论依据。【方法】以化学杂交剂SQ-1诱导的生理型雄性不育系ms(A)-西农1376及对应正常可育系(A)-西农1376为试材，用TRITC-phalloidin标记细胞内微丝，苯胺蓝标记胼胝质，qRT-PCR技术分别对肌动蛋白解聚因子TaADF（Actin depolymerizing factor）、类葡聚糖合成酶TaGSL（Glucan synthase-like）进行差异表达分析。【结果】(1)在减数分裂前期Ⅰ、中期Ⅰ、后期Ⅰ这三个时期，生理型雄性不育系花粉细胞的微丝结构与可育系没有显著差异：前期Ⅰ，微丝分布于整个细胞质中，细胞核区域也可见少量微丝环绕细胞核；中期Ⅰ，微丝分布在细胞质中，在形成纺锤体部位染色更深，形成纺锤体微丝，由细胞两极发出的纺锤体微丝伸向赤道板；后期Ⅰ，在向两极移动的染色体的中间部位染色较深，微丝分布较多。(2)在早末期Ⅰ，与可育系相比，不育系花粉细胞没有形成清晰且明显可见的中国灯笼状成膜体微丝结构，且在细胞中线部位亦没有清晰可见的微丝累积。(3)晚末期Ⅰ，可育系花粉细胞在形成细胞板的部位是线性的、平滑的，成膜体微丝消失，而不育系花粉细胞在形成细胞板的部位形成了很大的缝隙，同时，可育系胼胝质在细胞板处的沉积比较平滑，而不育系胼胝质在细胞板处的沉积较可育系相比缺乏，并且是褶皱的、有裂纹的。(4)四分体时期，可育系花粉可见围绕细胞核的辐射状微丝，不育系花粉细胞中微丝呈模糊状态，并且不育系中胼胝质染色的整体荧光强度较可育系减弱。利用实时荧光定量PCR技术分析肌动蛋白解聚因子TaADF和类葡聚糖合成酶TaGSL在减数分裂期的相对表达量，结果发现，不育系中TaADF的相对表达量是可育系的4.28倍，由于TaADF表达量上调，加剧了细胞内微丝解聚，微丝结构受到破坏，同时不育系中TaGSL表达量下降，只有可育系的0.83倍，胼胝质的沉积也受到影响。【结论】TaADF在不育系中上调表达，破坏了细胞内微丝的正常结构，使微丝不能正常行使其功能，进而可能导致花药发育中与育性相关的某些代谢通路等受到影响。与此同时，微丝结构的破坏导致细胞板形成出现异常也可能是引起胼胝质在细胞板处沉积受到影响的一个重要原因。因此，微丝和胼胝质的异常变化与化学杂交剂SQ-1诱导的生理型雄性不育密切相关。

关键词: 普通小麦, 生理型雄性不育, 减数分裂, 微丝骨架, 胼胝质

Abstract: 【Objective】The organization of actin filaments and callose and the expression of related genes in physiological male sterility wheat pollen cells were studied to reveal the relationship between actin filaments and callose and pollen abortion in physiological male sterility wheat. The results of the study will provide a theoretical basis for further study on the mechanism of male sterility induced by chemical hybridizing agents SQ-1 in wheat. 【Method】The physiological male sterility line ms(A)-Xinong1376 and corresponding normal fertile wheat (A)-xinong1376 were used as test materials. The TRITC-phalloidin was used to stain actin filaments and Aniline blue was used to stain callose. QRT-PCR was used to analyze the expression of TaADF (Actin depolymerizing factor) and TaGSL (Glucan synthase-like).【Result】At prophase I, metaphase I, and anaphase I, there were no significant differences between physiological male sterility and male fertility. At prophase I, actin filaments distributed in cytoplasm of the cell and there was also distribution of some actin filaments in nuclear zone. At metaphase I, actin filaments distributed in cytoplasm. They were stained deeper in the spindle position and formed actin spindle. At anaphase I, actin filaments between the poles of the chromosomes were stained deeper, so there were more actin filaments distributed there. At early telophase I, we observed that there were no sharp actin filaments at leading edges of phragmoplasts and the overlapped actin filaments at midline were obscure in physiological male sterility. At late telophase I, there was a cell plate at the midline of phragmoplasts, it was linear and smooth, but in the physiological male sterile line, the linear cell plate was not seen, the midzone of dyads was hollow. At the same time, the deposition of callose on the cell plate was insufficient and the cell plate was wrinkled and cleft. At tetrad, actin filaments were obscure and had no silky feeling and callose fluorescence was weaker in the physiological male sterile. Then qRT-PCR technique was performed to analyze the expression patterns of the TaADF gene and TaGSL gene in sterile and fertile anthers during meiosis. The results indicated that the transcript of TaADF in the treated anthers was 4.28 times higher than that of the control. It indicated that because of the increased expression of TaADF, the rate of dissociation of actin filaments was increased, so the microfilaments were obscure. The transcript of TaGSL in the treated anthers was 0.83 times lower than that of the control. Callose deposition was influenced by SQ-1. 【Conclusion】The increased expression level of TaADF in the physiological male sterility line destroyed the normal structure of actin filaments. The actin filaments could not execute their functions. Then it may influence certain metabolic pathways associated with fertility in anther development. At the same time, abnormal actin filaments caused abnormal cell plate formation which may have an effect on callose deposition. Therefore, it was estimated that it has a close relationship among the changes of actin filaments and callose and the physiological male sterility induced by SQ-1.

Key words: common wheat, physiological male sterile, meiosis, actin filaments, callose

张姣，朱启迪，巨岚，张改生，于永昂，牛娜，王军卫，马守才. 小麦生理型雄性不育系微丝骨架和胼胝质的变化与其相关基因的表达分析[J]. 中国农业科学, 2015, 48(14): 2687-2696.

ZHANG Jiao, ZHU Qi-di, JU Lan, ZHANG Gai-sheng, YU Yong-ang, NIU Na, WANG Jun-wei, MA Shou-cai. Organization Changes of Actin Cytoskeleton and Callose with the Expression Analysis of Related Genes in Physiological Male Sterile Wheat[J]. Scientia Agricultura Sinica, 2015, 48(14): 2687-2696.

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小麦生理型雄性不育系微丝骨架和胼胝质的变化与其相关基因的表达分析

Organization Changes of Actin Cytoskeleton and Callose with the Expression Analysis of Related Genes in Physiological Male Sterile Wheat

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