大豆 GmGolS1 的克隆及转基因烟草耐高温性鉴定
作者:
作者单位:

1.齐齐哈尔大学生命科学与农林学院;2.黑龙江省农业科学院畜牧兽医分院

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基金项目:

黑龙江省普通本科高等学校青年创新人才培养计划( UNPYSCT-2017153);齐齐哈尔大学研究生创新科研项目(YJSCX2020041)


Cloning of Soybean GmGolS1 and Identification of Heat Resistance in Transgenic Tobacco
Author:
Affiliation:

1.College of Life Science and Agro-Forestry,Qiqihar University;2.Branch of Animal Husbandry and Veterinary of Heilongjiang Academy of Agricultural Sciences

Fund Project:

Training Plan of Young Creative Talents of Average Four-year College of Heilongjiang(UNPYSCT-2017153),Innovative Research Project for Graduate Students of Qiqihar University(YJSCX2020041)

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    摘要:

    棉子糖系列寡糖(RFOs,raffinose family oligosaccharides)是植物体内一种重要的渗透调节物质,肌醇半乳糖苷合 成酶(GolS,galactinol synthase)是 RFOs 合成过程中的关键酶。本研究从大豆中克隆了 GmGolS1 基因。序列分析结果显 示,GmGolS1 基因位于大豆 3 号染色体,基因序列内部有 3 个内含子,开放阅读框(ORF,open reading frame)全长 1020 bp, 编码的多肽含有 339 个氨基酸,分子量 38.82 kDa,等电点 5.47。GmGolS1 氨基酸序列具有植物 GolS 蛋白的共同特征:一 个保守的丝氨酸磷酸化位点、一个锰离子结合相关的 DXD 元件和一个羧基末端疏水性五肽。蛋白系统进化分析结果显示, GmGolS1 与沙东青 AmGolS 和蒺藜苜蓿 MtGolS 的亲缘关系较近。实时荧光定量 PCR 结果显示 GmGolS1 在大豆幼苗中可 以不同程度的应答高温、低温、干旱及高盐胁迫,且高温胁迫下 GmGolS1 的表达量升高最明显。构建 GmGolS1 植物表达载 体并转化烟草,获得 4 个转基因烟草株系。GolS 酶活性检测结果显示,转基因烟草中 GmGolS1 转录后的产物发挥了 GolS 的催化活性。高温胁迫下 GmGolS1 转基因烟草的植株表型及电解质渗透率、可溶性糖含量和丙二醛含量的检测结果表明, GmGolS1 提高了转基因烟草的耐高温性。

    Abstract:

    Raffinose family oligosaccharides(RFOs)are one of important osmoregulatory substance in plants. Galactinol synthase(GolS)is a key enzyme in the synthesis of RFOs. In this study,GmGolS1 was cloned from soybean based on sequence homology. GmGolS1 was found on chromosome 3 with three introns. The full length open reading frame(ORF)of GmGolS1 was 1020 bp,which encodes 339 amino acids with the molecular weight of 38.82 kDa,and the isoelectric point of 5.47. The amino acid sequence GmGolS1 has characteristics that are commonly detected in plant GolS proteins:a conserved serine phosphorylation site,a manganese ion binding associated DXD element,and a carboxy-terminal hydrophobic pentapeptide. The phylogenetic analysis showed that GmGolS1 was closely related to AmGolS and MtGolS. Real-time fluorescence quantitative PCR analysis showed that GmGolS1 was inducible upon heat,low temperature,drought and salt stresses in different degrees in seedlings,and the most abundant expression was observed under heat stress treatment. By transforming GmGolS1 was into tobacco,four transgenic lines were identified using genomic DNA PCR and real-time fluorescence quantitative PCR. Test for GolS enzyme activity showed that the transcribed product of GmGolS1 in transgenic tobacco played the catalytic activity of GolS. Gained from plant phenotype,electrolyte leakage,soluble carbohydrate content and malondialdehyde content of GmGolS1 transgenic tobacco under heat stress,GmGolS1 was found correlating with improved heat resistance in transgenic tobacco.

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李铭杨,邱 爽,何佳琦,等.大豆 GmGolS1 的克隆及转基因烟草耐高温性鉴定[J].植物遗传资源学报,2022,23(2):575-582.

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  • 收稿日期:2021-08-23
  • 最后修改日期:2021-09-13
  • 录用日期:2021-09-28
  • 在线发布日期: 2022-03-10
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