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| 微管蛋白β-Tubulin在二、三倍体虹鳟(Oncorhynchus mykiss)表达与定位分析 |
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黄天晴1, 史秀兰1, 王炳谦1, 邹作宇2, 谷伟1, 刘恩慧1, 程琳1, 刘洋1, 董福霖1, 潘赞宇1, 徐革锋1
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1.中国水产科学研究院黑龙江水产研究所 农业农村部淡水水产生物技术与遗传育种重点实验室 哈尔滨 150070;2.哈尔滨市农业科学院 哈尔滨 150029
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| 摘要: |
| 微管构成纺锤体主要架构，在染色体配对与分离过程起重要作用。通过β-tubulin基因克隆，实时荧光定量，Western blot及免疫组织化学技术对三倍体虹鳟卵巢败育根本原因进行探究。结果表明β-tubulin基因开放阅读框1 533 bp，编码424个氨基酸。β-Tubulin蛋白质的相对分子量为47.5 kDa。虹鳟（Oncorhynchus mykiss）与银大马哈鱼（O.kisutch）同源性高达99.59%。系统发育进化树显示，虹鳟与大鳞大马哈鱼（O.tshawytscha）和红点鲑（Salvelinus alpinus）聚为一支。实时定量检测发现β-tubulin基因在二倍体雌性虹鳟卵巢表达量极显著高于其他组织。在240-330 dpf同一发育阶段中，β-tubulin基因在二倍体雌性虹鳟卵巢中的表达量较三倍体雌性虹鳟相对较高，且均存在极显著差异。免疫组化结果显示，240-330 dpf，β-Tubulin蛋白主要在二倍体虹鳟卵母细胞核内表达，在三倍体虹鳟卵原细胞核内表达量较少。推测三倍体虹鳟性腺败育可能与β-tubulin基因的低表达与组织异常定位有关，结果为进一步揭示三倍体虹鳟卵母细胞减数分裂受阻提供基础理论。 |
| 关键词: 虹鳟 β-tubulin 基因克隆 组织表达分布 |
| DOI：10.11693/hyhz20210300064 |
| 分类号: |
| 基金项目:财政部和农业农村部：国家现代农业产业技术体系资助，CARS-46号；中国水产科学研究院基本科研业务费项目，2020TD32号；中国水产科学研究院基本科研业务费项目，2018HY-ZD0302号。 |
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| ANALYSIS THE EXPRESSION AND LOCALIZATION OF β-TUBULIN IN DIPLOID AND TRIPLOID RAINBOW TROUT (ONCORHYNCHUS MYKISS) |
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HUANG Tian-Qing1, SHI Xiu-Lan1, WANG Bing-Qian1, ZOU Zuo-Yu2, GU Wei1, LIU En-Hui1, CHENG Lin1, LIU Yang1, DONG Fu-Lin1, PAN Zan-Yu1, XU Ge-Feng1
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1.Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Freshwater Aquatic Biotechnology and Breeding, Ministry of Agriculture and Rural Affairs, Harbin 150070, China;2.Harbin Academy of Agricultural Sciences, Harbin 150029, China
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| Abstract: |
| Microtubules constitute the main structure of the spindle and play an important role in chromosome pairing and separation. To explore the critical reasons of ovarian abortion in triploid rainbow trout and analyze the expression and localization of β-tubulin, the β-tubulin gene was cloned, and real-time PCR and the Western blot and immunohistochemistry were performed. Results show that the β-tubulin gene had an open reading frame of 1533 bp encoding 424 amino acids. The relative molecular weight of β-Tubulin protein was 47.5 kDa. The β-tubulin sequence of rainbow trout Oncorhynchus mykiss shared the highest homology with O. kisutch. The phylogenetic tree indicated that β-Tubulin of O. mykiss was associated with O. tshawytscha and Salvelinus alpinus. The expression of β-tubulin gene in diploid female rainbow trout gonald was significantly higher than that in other tissues. The expression of β-tubulin gene in the ovaries of diploid female rainbow trout was relatively higher than that of triploid female rainbow trout in the same development stage, and there were significant differences between diploid and triploid female rainbow trout from 240 to 330 dpf (days post fertilization). Immunohistochemical results showed that β-Tubulin protein was mainly expressed in the oocyte nucleus of diploid rainbow trout from 240 to 330 dpf, but less in the oocyte nucleus of triploid rainbow trout. Therefore, we believed that the abortive ovary of triploid rainbow trout was related to the low expression of β-tubulin gene and abnormal tissue location. This study provided a basic theory for further exploring the effect of polyploidy on gonadal development. |
| Key words: Oncorhynchus mykiss β-tubulin gene cloning tissue expression distribution |