紫花苜蓿F-box蛋白基因MsFTL的克隆及功能分析
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黑龙江省分子细胞遗传与遗传育种重点实验室/哈尔滨师范大学生命科学与技术学院,哈尔滨150000

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国家自然科学基金(31470571和31770575);国家高技术研究发展计划项目(2013AA102607);黑龙江省重大攻关 目(GA18B104)和国家转基因生物新品种培育重大专项(2016ZX08004-002),国家自然科学基金项目(面上项目,重点项目,重大项目)


Cloning and Functional Analysis of F-box Protein Gene MsFTL in Alfalfa( Medicago sativa L)
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Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province/ College of Life1Science and Technology, Harbin Normal University,Harbin 150000

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National Natural Science Foundation of China(31470571); Supported by National High Technology Research and Development Program of China(2013AA102607); Major Research Project in Heilongjiang Province(GA15B105); National Major Projects of Transgenic Organisms Breeding New Varieties(2016ZX08004-002)

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    摘要:

    FTL(F-box Triple LRR protein)是F-box 蛋白家族的成员,具有F-box 保守结构域,在植物抵御逆境胁迫 过程中起重要作用。本研究参考低温胁迫下紫花苜蓿转录组数据设计引物,通过RT-PCR 克隆获得紫花苜蓿MsFTL 基因, 该基因的全长1422 bp,编码473 个氨基酸。该蛋白含有一个F-box 结构域及3 个LRR 重复。系统进化分析表明,MsFTL 与蒺藜苜蓿XP_003626345.1 F-box/FBD/LRR-repeat protein 亲缘关系最近。两者蛋白序列比对发现共有11 个差异位点。 在低温、盐、干旱以及外源ABA 处理下,MsFTL 基因受到诱导,表达量上调。构建植物过表达载体pCBM-MsFTL,通过农 杆菌介导法转化烟草。对经过抗性筛选、PCR 和Real-time PCR 验证的转基因植株进行低温抗性鉴定。在-4℃低温胁迫 下,野生型烟草叶片出现了明显的萎蔫失水现象,而转基因烟草萎蔫程度相对较轻。生理检测结果表明,4℃处理24 h 之后,转基因烟草的可溶性蛋白含量、可溶性糖含量、SOD 活性,CAT 活性高于野生型,MDA 含量低于野生型。本研究表 明,MsFTL 基因在提高植物对低温胁迫的抗性方面具有重要的作用。

    Abstract:

    FTL (F-box Triple LRR ) protein belongs to the family of F-box proteins that play an important role in tolerance to low temperature. In this study, an MsFTL gene was isolated by candidate gene approach from alfalfa, and this gene was differently expressed in leaf under cold stress. The full-length cDNA of MsFTL gene was 1422 bp, which putatively encoded for 473 amino acids. By bioinformatic analysis, the MsFTL protein was found to carry an F-box domain and three LRR repeats in C-terminus. MsFTL showed homology closely to XP_003626345.1, a member of F-box/FBD/LRR-repeat protein in Medicago truncatula, with 11 differential sites between the two proteins. The expression of MsFTL was induced by low temperature, salt, drought stress and ABA treatments. We generated the transformation construct and transformed the MsFTL into tobacco. Under -4°C, wild-type tobacco leaves showed remarkable wilting, while the transgenic plants presented slightly stressed phenotype.Transgenic plants accumulated higher content of soluble protein and soluble sugar than that of wild-type. The activity of SOD and CAT in transgenic plants was higher than those of wild type under low temperature (4°C) for 24 h. Moreover, the MDA content in transgenic plants was lower than that of wild type. Thus, these results suggested that overexpressing MsFTL might significantly improve tobacco plant tolerance to low temperature stress.

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陈秀秀,张 彤,余倩文,等.紫花苜蓿F-box蛋白基因MsFTL的克隆及功能分析[J].植物遗传资源学报,2019,20(3):750-759.

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  • 收稿日期:2018-09-29
  • 最后修改日期:2019-03-15
  • 录用日期:2018-11-12
  • 在线发布日期: 2019-05-13
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