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中国油料作物学报 ›› 2021, Vol. 43 ›› Issue (4): 638-.doi: 10.19802/j.issn.1007-9084.2020071

• 遗传育种 • 上一篇    下一篇

大豆GmPUB32 基因的克隆及耐盐功能分析

苌兴超(1994-),男,硕士,研究方向为作物遗传育种,E-mail: changxingchao1213@163.com   

  1. 东北农业大学大豆生物学教育部重点实验室/农业农村部东北大豆生物学与遗传育种重点实验室,黑龙江哈尔滨,150030
  • 出版日期:2021-08-28 发布日期:2021-08-30
  • 通讯作者: 李永光(1984-),男,副研究员,研究方向为大豆遗传育种及生物技术应用,E-mail: yongguangli@neau.edu; 李文滨(1958-),男,教授,研究方向为作物遗传育种,E-mail: wenbinli@yahoo.com
  • 基金资助:
    转基因生物新品种培育科技重大专项任务(2016ZX08004002)

Cloning and salt resistance analysis of soybean GmPUB32 gene

  1. Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education/Key Labora⁃
    tory of Soybean Biology and Genetics Breeding of Ministry of Agriculture and Rural Affairs, Harbin 150030, China
  • Online:2021-08-28 Published:2021-08-30

摘要: 泛素化系统通过介导蛋白质的翻译后修饰,参与包括细胞分化、激素应答、生物与非生物胁迫响应等多个生物学过程,其中含有U-box基因的泛素连接酶为泛素化系统的重要酶之一。课题组前期研究发现大豆Gly⁃ma.13G115900(GmPUB32)基因编码一个泛素连接酶(PUB),其表达量在根中受盐胁迫影响显著。本研究在大豆品种垦丰16中克隆得到大豆U-box家族基因GmPUB32,序列分析结果表明GmPUB32 基因的开放阅读框长度为513bp,编码170个氨基酸,在其83-131个氨基酸之间含有一个RING/U-box保守结构域;亚细胞定位分析结果显示Gm⁃PUB32蛋白定位于细胞膜与细胞质中;GmPUB32 基因在大豆根、茎、叶片与荚果中均能检测到不同程度的表达,在根中表达量最高,GUS组织化学染色进一步明确其在根中发挥主要作用;荧光定量PCR分析结果显示,在200 mmol/L NaCl处理后,与对照相比随着处理时间的延长GmPUB32 在根中的表达量呈现出显著的下降趋势,表明GmPUB32可能参与大豆对于盐胁迫的应答过程;通过对过表达GmPUB32 的T3拟南芥的盐胁迫耐受性进行分析,结果表明转基因拟南芥的萌发率、子叶绿化率与盐胁迫下的存活率均明显低于野生型,此外,GmPUB32 过表达转基因毛状根的生长速率相比野生型也明显受到抑制。由此推断,GmPUB32 可能作为负调控因子参与大豆对于盐胁迫的应答过程,降低植物对于盐胁迫的耐受性。

关键词: 盐胁迫, 大豆, U-box, 拟南芥

Abstract:

Ubiquitination system is involved in many biological processes, including cell differentiation, hormone response, biological and abiotic stress response, by mediating the post-translational modification of proteins.The ubiquitination ligase containing U-box gene is one of the important enzymes in the Ubiquitination system. In this study, soybean U-box family gene Glyma. 13G115900 (GmPUB32) was cloned from soybean variety Kengfeng 16. Sequence analysis showed that GmPUB32 gene has an open reading frame with a length of 513 bp, encoding 170 amino acid with a RING/ U-box conserved domain between its 83-131 amino acids. The results of subcellular localization analysis showed that GmPUB32 protein was located in the cell membrane and cytoplasm. The expression of GmPUB32 gene in soybean root, stem, leaf and pod was detected on different levels, and it is the highest in root. Fluorescence quantitative PCR analysis showed that the expression of GmPUB32 was significantly down-regulated under salt stress. Salt stress tolerance of T3 generation of Arabidopsis thaliana overexpressing GmPUB32 were analyzed, and the results showed that the germination rate, cotyledon afforestation rate and survival rate were significantly lower than that of the wild type under salt stress, in addition, growth rate of transgenic hairy root overexpressing GmPUB32 also significantly was suppressed compared with the wild type. Therefore, GmPUB32 may play a role in response to salt stress process as a negative regulatory factors.

Key words: salt stress, soybean, U-box, Arabidopsis

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