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    冯佳琳, 刘聪聪, 戚相玉, 邸泽鑫, 鲁仪增, 郑健. 花楸树热激蛋白SpHSP70-3基因克隆与功能分析[J]. 北京林业大学学报, 2022, 44(9): 52-61. DOI: 10.12171/j.1000-1522.20210235
    引用本文: 冯佳琳, 刘聪聪, 戚相玉, 邸泽鑫, 鲁仪增, 郑健. 花楸树热激蛋白SpHSP70-3基因克隆与功能分析[J]. 北京林业大学学报, 2022, 44(9): 52-61. DOI: 10.12171/j.1000-1522.20210235
    Feng Jialin, Liu Congcong, Qi Xiangyu, Di Zexin, Lu Yizeng, Zheng Jian. Cloning and functional analysis of heat shock protein SpHSP70-3 gene from Sorbus pohuashanensis[J]. Journal of Beijing Forestry University, 2022, 44(9): 52-61. DOI: 10.12171/j.1000-1522.20210235
    Citation: Feng Jialin, Liu Congcong, Qi Xiangyu, Di Zexin, Lu Yizeng, Zheng Jian. Cloning and functional analysis of heat shock protein SpHSP70-3 gene from Sorbus pohuashanensis[J]. Journal of Beijing Forestry University, 2022, 44(9): 52-61. DOI: 10.12171/j.1000-1522.20210235

    花楸树热激蛋白SpHSP70-3基因克隆与功能分析

    Cloning and functional analysis of heat shock protein SpHSP70-3 gene from Sorbus pohuashanensis

    • 摘要:
        目的  探讨热激蛋白(HSP)在花楸树响应高温胁迫过程中的作用,以期为花楸树引种低海拔地区提供理论基础。
        方法  以2 ~ 4年生花楸树实生苗为研究对象,进行了花楸树SpHSP70-3基因的克隆、系统进化分析、组织特异性表达模式以及响应高温胁迫的表达机制研究,并利用农杆菌介导法转化拟南芥,对SpHSP70-3基因在高温胁迫下的响应进行了异源验证。
        结果  SpHSP70-3基因开放阅读框全长为2 088 bp,编码695个氨基酸;SpHSP70-3蛋白与蔷薇科梨属的白梨PbHSP70同源性最高。内源性表达分析显示:SpHSP70-3基因在叶片中表达量最高,在花蕾、初花、盛花时表达量偏低;42 ℃处理花楸树后,发现前6 h SpHSP70-3基因表达量没有显著变化,12 h时表达量增至对照组的12倍,24 h时表达倍数最高,为对照组的19倍。对3个转SpHSP70-3基因拟南芥纯合株系(OE1、OE2、OE3)和野生型拟南芥(WT)进行45 ℃高温处理后,OE1、OE2、OE3中的丙二醛(MDA)含量均高于WT,且过氧化氢酶(CAT)活性和过氧化物酶(POD)活性结果均低于WT。此外,SpHSP70-3在转基因株系中的表达量随着处理时间的增加而上升,并且其抑制了正调节因子AtHSP70、AtHSP18.2、AtHsfA1DAtHsfA1A的表达,同时诱导了负调节因子AtHsfB2B的上调表达。
        结论  SpHSP70-3在花楸树响应高温胁迫过程中起负调控作用,初步推测SpHSP70-3是花楸树引种低海拔地区响应高温响胁迫机制中的负调控因子。

       

      Abstract:
        Objective  This paper aims to explore the function of heat shock protein (HSP) in Sorbus pohuashanensis in response to high temperature stress, and to provide theoretical basis for the introduction of species in low altitude areas.
        Method  Taking 2−4 years old seedlings of Sorbus pohuashanensis as the research object, the cloning, phylogenetic analysis, tissue-specific expression pattern and expression mechanism in response to high temperature stress of Sorbus pohuashanensis were studied, and the response function of transgenic Arabidopsis thaliana to high temperature stress was verified by agrobacterium-mediated transformation.
        Result  The full-length open reading frame of SpHSP70-3 gene was 2 088 bp, encoding 695 amino acids. SpHSP70-3 protein had the highest homology with Pyrus bretschneideri PbHSP70 in Rosaceae. Endogenous expression analysis showed that the expression of SpHSP70-3 gene was the highest in leaves, but the expression was low in bud, first flower and full flower. The expression level did not change significantly 6 h before treatment at 42 ℃, but increased to 12 times of the control group at 12 h, and reached the highest level at 24 h, but only 19 times of the control group. After three homozygous arabidopsis lines of OE1, OE2 and OE3 with SpHSP70-3 gene and WT arabidopsis were treated at 45 ℃, the MDA contents in OE1, OE2 and OE3 were all higher than WT, and the CAT enzyme activity and POD enzyme activity were lower than WT. In addition, the expression of SpHSP70-3 in transgenic lines increased with the increase of treatment time, and inhibited the expression of positive regulatory factors AtHSP70, AtHSP18.2, AtHsfA1D and AtHsfA1A, while induced the up-regulation of negative regulatory factor AtHsfB2B.
        Conclusion  SpHSP70-3 plays a negative regulatory role in the response of Sorbus pohuashanensis to high temperature stress, and preliminarily speculates that SpHSP70-3 is a negative regulatory factor in the response mechanism of Sorbus pohuashanensis to high temperature stress at low altitude.

       

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