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园艺学报 ›› 2019, Vol. 46 ›› Issue (12): 2323-2334.doi: 10.16420/j.issn.0513-353x.2019-0126

• 研究论文 • 上一篇    下一篇

柑橘雄性不育类蛋白基因MSLP的克隆与分析

邱文明1,张祖铭2,徐育海1,何秀娟1,孙中海1,仝 铸1,肖 翠1,吴黎明1,郭文武2,*   

  1. 1湖北省农业科学院果树茶叶研究所,湖北省农业科技创新中心果树茶叶研究分中心,武汉 430064;2华中农业大学园艺植物生物学教育部重点实验室,园艺林学学院,武汉 430070
  • 出版日期:2019-12-25 发布日期:2019-12-25
  • 基金资助:
    国家自然科学基金项目(31501731,31530065);湖北省农业科学院竞争性计划项目(2016jzxjh028);湖北省农业科技创新中心资助项目(2016-620-000-001-031)

Cloning and Functional Analysis of Male Sterile 2-like Protein Gene(MSLP)in Citrus

QIU Wenming1,ZHANG Zuming2,XU Yuhai1,HE Xiujuan1,SUN Zhonghai1,TONG Zhu1,XIAO Cui1,WU Liming1,and GUO Wenwu2,*   

  1. 1Institute of Fruit and Tea,Hubei Academy of Agricultural Sciences,Fruit and Tea Subcenter of Hubei Innovation Center of Agricultural Science and Technology,Wuhan 430064,China;2Key Laboratory of Horticultural Plant Biology (Ministry of Education),College of Horticulture and Forestry Sciences,Huazhong Agricultural University,Wuhan 430070,China
  • Online:2019-12-25 Published:2019-12-25

摘要: 为分析柑橘雄性不育类蛋白基因MSLP的功能,利用RT-PCR法在6个典型柑橘品种中克隆了MSLP cDNA全长,检测到4种不同的转录本,其中MSLP-B型(1 476 bp)在6个品种中均能克隆到。序列和系统进化分析表明,MSLP包含3个脂酰辅酶A还原酶(fatty acyl-CoA reductase,FAR)C端结构域,MSLP-B与甜橙FAR1-like蛋白和克里曼丁橘FAR3蛋白聚在一类,与蔷薇科植物如碧桃、月季、甜樱桃和梅的FAR蛋白亲缘关系最近;MSLP在柑橘基因组有5 ~ 8个拷贝数,MSLP-B作用于细胞质,在无核椪柑各时期花器官中的表达量均高于普通有核椪柑;MSLP-B转基因烟草表型分析表明,转基因植株成熟期花柱/花丝比值高于野生型,尽管花粉活力不受影响,但不饱满的椭圆形花粉粒显著增多。

关键词: 柑橘, 雄性不育, MSLP, 无核

Abstract: To elucidate the functions of male sterile 2-like protein(MSLP)in citrus,the full-length cDNAs of MSLP were cloned in six typical citrus varieties using RT-PCR. Four transcripts of MSLP were identified,and only MSLP-B existed in all varieties. Structure analysis showed MSLP contained three conserved fatty acyl-CoA reductase(FAR)C terminal domains. Phylogenetic analysis suggested that MSLP-B clustered with sweet orange FAR-like protein and clementine FAR3 protein,and they had closer genetic relationship with FAR-proteins in rosaceous plant like peach,rose,sweet cherry and plum. Southern blotting showed there were 5–8 copies of MSLP in citrus. Transient expression of MSLP-B-GFP in Nicotiana benthamiana leaves indicated it functioned in cytoplasm. qRT-PCR results showed the expression of MSLP-B in floral organs of seedless Ponkan was up-regulated compared with common seedy Ponkan. Over-expression of MSLP-B in tobacco showed that the ratio of stylar length to filament length in mature flower was higher than non-transgenic tobacco. The viability of pollen grains was normal in transgenic lines. However,the number of unsaturated elliptic pollen grains increased significantly.

Key words: citrus, male sterility, male sterile 2-like protein, seedlessness

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