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花楸树热激蛋白SpHSP70-3基因克隆与功能分析

冯佳琳 刘聪聪 戚相玉 邸泽鑫 鲁仪增 郑健

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. 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. 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. 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. 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

doi: 10.12171/j.1000-1522.20210235
基金项目: 国家自然科学基金项目(31770369)。
详细信息
    作者简介:

    冯佳琳。研究方向:林木种质资源与育种。Email:2628493430@qq.com 地址:102206北京市昌平区回龙观镇北农路7号北京农学院园林学院

    责任作者:

    郑健,教授。研究方向:林木种质资源与育种。Email:buazhengjian708@126.com 地址:同上

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。

     

  • 图  1  SpHSP70-3基因的PCR扩增条带

    M. marker;(1)、(2)分别为SpHSP70-3基因的cDNA和gDNA的PCR扩增条带。(1)、(2) meanPCR amplified bands of cDNA and gDNA of SpHSP70-3 gene,respectively.

    Figure  1.  PCR amplification pattern of SpHSP70-3 gene

    图  2  SpHSP70-3基因cDNA序列及其编码蛋白保守基序

    ▭、—、▲表示核苷酸结合位点、核苷酸互换因子互换位点、底物结合域。▭、—、▲ indicates nucleotide binding sites、 nucleotide exchange factor exchange sites and substrate binding domains.

    Figure  2.  cDNA sequence and its conserved coding protein domain of SpHSP70-3

    图  3  SpHSP70-3蛋白与其他植物HSP70的同源序列比对

    Cm. 南瓜Cucurbita moschata (XP_022930091.1);Cq. 藜麦Chenopodium quinoa (XP_021743068.1);Cs. 黄瓜Cucumis sativus (NP_001295865.1);Fv. 草莓Fragaria vesca subsp. Vesca (XP_004300629.1);Md. 苹果Malus domestica (XP_028963255.1);Pb. 白梨Pyrus bretschneideri (XP_009353782.1);Pa. 甜樱桃Prunus avium (XP_021815855.1);Pd. 扁桃Prunus dulcis (XP_034211692.1);Rc. 月季Rosa chinensis (XP_024170474.1);Pe. 胡杨Populus euphratica (XP_011016196.1);Vr. 葡萄Vitis riparia (XP_034673011.1);NBD. DNA结合域 Nucleotide binding domain;SBD. 底物结合域 Substrate binding domain.

    Figure  3.  Homologous alignment of SpHSP70-3 protein with HSP70 protein in other plants

    图  4  SpHSP70-3蛋白与其他植物HSP70的系统进化树

    Figure  4.  Phylogenetic tree of SpHSP70-3 protein with HSP70 protein in other plants

    图  5  花楸树SpHSP70-3基因的内源性表达分析

    a. SpHSP70-3基因组织特异性表达;b. 42 ℃高温胁迫下SpHSP70-3基因表达分析。YL. 幼叶;ML. 成叶;B. 花蕾;IA. 初花;MA. 盛花;EA. 末花;F. 果;S. 茎;R. 根。不同小写字母表示差异显著,P < 0.05。下同。a, Tissue specific expression of SpHSP70-3; b, Relative expression of SpHSP70-3 at 42 ℃ treatment . YL, younger leaf; ML, mature leaf; B, bud; IA, initial anthesis; MA, mature anthesis; EA, end anthesis; F, fruit; S, stem; R, Root. Different letters represent significant differences at P < 0.05 level. The same below.

    Figure  5.  Endogenous expression analysis of SpHSP70-3 gene in Sorbus pohuashanensis

    图  6  45 ℃胁迫下转SpHSP70-3基因拟南芥基因表达分析

    a. 转基因拟南芥OE1、OE2、OE3在45 ℃下的SpHSP70-3基因表达; b. 正常条件下株系OE3和WT中基因表达分析; c. 45 ℃处理下株系OE3和WT中基因表达分析。*代表P < 0.05平上的显著差异,**代表在P < 0.01水平上的显著差异,下同。a, Expression analysis of SpHSP70-3 gene in transgenic Arabidopsis thaliana under 45 ℃ stress; b, Analysis of target gene expression in OE3 and WT under normal conditions; c, Analysis of target gene expression in OE3 and WT under 45 ℃ stress. * represent significance at P < 0.05, ** represent significance at P < 0.01. The same below.

    Figure  6.  Expression analysis of genes in transgenic Arabidopsis thaliana under 45 ℃ stress

    图  7  45 ℃胁迫下转SpHSP70-3基因和野生型拟南芥的表型及生理指标分析

    a. 转SpHSP70-3基因拟南芥OE1、OE2、OE3株系在45 ℃下的胁迫表型;b. MDA含量分析;c. CAT酶活性分析;d. POD酶活性分析。a, Phenotype of transgenic Arabidopsis thaliana with SpHSP70-3 gene under 45 ℃ stress; b, Analysis of MDA content; c, Analysis of CAT activity; d, Analysis of POD activity.

    Figure  7.  Phenotypic and physiological indexes analysis of transgenic SpHSP70-3 gene and wild Arabidopsis thaliana under 45 ℃ stress

    表  1  引物序列

    Table  1.   Primer sequence.

    引物 Primer name序列(5′-3′) Sequence ( 5′-3′ )引物 Primer name序列(5′-3′) Sequence ( 5′-3′ )
    SpHSP70-3-F ATGGCTTCCGCGCAAA SpHSP70-3-R CTCGCTCACCTGCTGTCG
    SpHSP70-3-TF CACCATGGCTTCCGCGCAAA SpHSP70-3-TR CTCGCTCACCTGCTGTCG
    Spβ-actin-QF TGGATGGCTGGAAGAGGA Spβ-actin-QR GAGCGGGAAATTGTGAGG
    SpHSP70-3-QF TCTCTTCTCCTTGTCCTCCTG SpHSP70-3-QR TTCTATCCGTCGCTGCTGT
    AtHSP70-QF ACTTGCTTATGAGTCTGAGGGTA AtHSP70-QR GCCTTGATAGGTGCTGATAGA
    AtHSP90-QF GGGGATTTGAACCTTATTGGA AtHSP90-QR CTGGCTGTCATCATTGTGCTT
    AtHSP18.2-QF CCGTTCTCGCAAGACTTATGG AtHSP18.2-QR CGGCGTTTCCTTCCAATCCAC
    AtHsfA1D-QF AGAAGCAACCGAGAACTGTAT AtHsfA1D-QR AGTAATGGACTAGAACCTCCC
    AtHsfA1A-QF TGGAGTCCGACGAACAATAGC AtHsfA1A-QR GGCGAACAAAGCTGGAGAAAT
    AtHsfB2B-QF AGTAGTGGATGTGGTGCTGGTG AtHsfB2B-QR CGAGATCAATTCGTCGTAAACC
    下载: 导出CSV
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    [43] 姚紫薇, 孙婧靓, 刘建祥, 等. 拟南芥热激转录因子HSFB2b负调控植物热形态建成[J/OL]. 浙江大学学报(农业与生命科学版), 2022. [2022-07-13]. http://kns.cnki.net/kcms/detail/33.1247.S.20220410.1723.002.Html.

    Yao Z W, Sun J L, Liu J X, et al. Heat shock transcription factor HSFB2b of Arabidopsis thaliana negatively regulates plant thermomorphogenesis. [J/OL]. Journal of Zhejiang University (Agriculture and Life Sciences ), 2022 [2022-07-13]. http://kns.cnki.net/kcms/detail/33.1247.S.20220410.1723.002.Html.
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出版历程
  • 收稿日期:  2021-06-21
  • 录用日期:  2022-07-18
  • 修回日期:  2021-07-27
  • 网络出版日期:  2022-07-22

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