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、AtHsfA1D和AtHsfA1A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 In order 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 is 2 088 bp, encoding 695 amino acids. SpHSP70-3 protein has 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 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 played a negative regulatory role in the response of Sorbus pohuashanensis to high temperature stress, and preliminarily speculated that SpHSP70-3 was a negative regulatory factor in the response mechanism of Sorbus pohuashanensis to high temperature stress at low altitude. -
Key words:
- Sorbus pohuashanensis /
- high temperature stress /
- heat shock protein 70 /
- gene cloning
-
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
-
[1] Wang W, Vinocur B, Shoseyo O, et al. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response[J]. Trends in Plant Science, 2004, 9(5): 244−252. doi: 10.1016/j.tplants.2004.03.006 [2] 黄祥富, 黄上志, 傅家瑞. 植物热激蛋白的功能及其基因表达的调控[J]. 植物学通报, 1999, 16(5): 530−536.Huang X F, Huang S Z, Fu J R. The function of plant heat shock protein and its gene expression regulation[J]. Chinese Bulletin of Botany, 1999, 16(5): 530−536. [3] Cho E K, Choi Y J. A nuclear-localized HSP70 confers thermoprotective activity and drought-stress tolerance on plants[J]. Biotechnology Letters, 2009, 31(4): 597−606. doi: 10.1007/s10529-008-9880-5 [4] Wang X Q, Yang P F, Gao Q, et al. Proteomic analysis of the response to high-salinity stress in Physcomitrella patens[J]. Planta, 2008, 228(1): 167−177. doi: 10.1007/s00425-008-0727-z [5] Tukaj S, Bisewska J, Roeske K, et al. Time- and dose-dependent induction of HSP70 in Lemna minor exposed to different environmental stressors[J]. Bulletin of Environmental Contamination & Toxicology, 2011, 87(3): 226−230. [6] Montero-Barrientos B, Hermosa R, Cardoza R E, et al. Transgenic expression of the Trichoderma harzianum hsp70 gene increases Arabidopsis resistance to heat and other abiotic stresses[J]. Journal of Plant Physiology, 2010, 167(8): 659−665. doi: 10.1016/j.jplph.2009.11.012 [7] Efremova S M. , Margulis B A., Guzhova I V., et al. Heat shock protein Hsp70 expression and DNA damage in Baikalian sponges exposed to model pollutants and wastewater from Baikalsk Pulp and Paper Plant[J]. Aquatic Toxicology, 2002, 57(4): 267−280. doi: 10.1016/S0166-445X(01)00209-0 [8] Hu C, Lin S Y, Charng C. Recent Gene Duplication and subfunctionalization produced a mitochondrial GrpE, the nucleotide exchange factor of the Hsp70 complex, specialized in thermotolerance to chronic heat stress in Arabidopsis[J]. Plant Physiology, 2012, 158(2): 747−758. doi: 10.1104/pp.111.187674 [9] Frederic A, Carole L. , Carsten L, et al. Virus induction of heat shock protein 70 reflects a general response to protein accumulation in the plant cytosol[J]. Plant Physiology, 2005, 138(1): 529−536. doi: 10.1104/pp.104.058958 [10] 王明强, 张道远. 植物热激蛋白70基因家族及其生物学功能研究进展[J]. 基因组学与应用生物学, 2015, 2: 421−428. doi: 10.13417/j.gab.034.000421Wang M Q, Zhang D Y. Research progress of plant heat shock protein 70 gene family and its biological function[J]. Genomics and Applied Biology, 2015, 2: 421−428. doi: 10.13417/j.gab.034.000421 [11] Jung K H, Ko H J, Nguyen M X, et al. Genome-wide identification and analysis of early heat stress responsive genes in rice[J]. Journal of Plant Biology, 2012, 55(6): 458−468. doi: 10.1007/s12374-012-0271-z [12] Su P H, Li H-m. Arabidopsis stromal 70-kDa heat shock proteins are essential for plant development and important for thermotolerance of germinating seeds[J]. Plant Physiology, 2008, 146(3): 1231−1241. doi: 10.1104/pp.107.114496 [13] Batcho, Sarwar, Tariq, et al. Identification and characterisation of heat shock protein gene (HSP70) family and its expression in Agave sisalana under heat stress[J]. The Journal of Horticultural Science and Biotechnology, 2020, 95(4): 1−13. [14] Vitale A, Bielli A, Ceriotti A. The binding protein associates with monomeric phaseolin[J]. Plant Physiology, 1995, 107: 1411−1418. doi: 10.1104/pp.107.4.1411 [15] 郑勇奇, 郑健, 张川红. 花楸树−城市绿化的新贵[J]. 中国城市林业, 2008, 6(2): 74−76. doi: 10.3969/j.issn.1672-4925.2008.02.024Zheng Y Q, Zheng J, Zhang C H. Sorbus pohuashanens is the upstart of urban greening[J]. Journal of Chinese Urban Forestry, 2008, 6(2): 74−76. doi: 10.3969/j.issn.1672-4925.2008.02.024 [16] 张向布. 花楸树−城市绿化的新贵[J]. 现代园艺, 2020(6): 133−134. doi: 10.3969/j.issn.1006-4958.2020.06.082Zhang X B. Sorbus pohuashanens isthe upstart of urban greening[J]. Xiandai Horticulture, 2020(6): 133−134. doi: 10.3969/j.issn.1006-4958.2020.06.082 [17] 郑健, 郑勇奇, 吴超, 等. 花楸树的地理分布及天然更新方式[J]. 林业科学, 2007, 43(12): 86−93. doi: 10.3321/j.issn:1001-7488.2007.12.015Zheng J, Zheng Y Q, Wu C, et al. The geographical distribution and natural regeneration of Sorbus pohuashanensis[J]. Scientia Silvae Sinicae, 2007, 43(12): 86−93. doi: 10.3321/j.issn:1001-7488.2007.12.015 [18] 李莉娜, 陈兴玲. 盐碱胁迫对花楸树的影响[J]. 吉林林业科技, 2018, 47(6): 10−12. doi: 10.16115/j.cnki.issn.1005-7129.2018.06.003Li L N, Chen X L. The effect of salt-alkali stress on Sorbus pohuashanensis[J]. Journal of Jilin Forestry Science and Technology, 2018, 47(6): 10−12. doi: 10.16115/j.cnki.issn.1005-7129.2018.06.003 [19] 郑健, 郑勇奇, 张川红, 等. 花楸树天然群体的遗传多样性研究[J]. 生物多样性, 2008, 16(6): 562−569. doi: 10.3321/j.issn:1005-0094.2008.06.006Zheng J, Zheng Y Q, Zhang C H, et al. Study on genetic diversity of natural population of Sorbus pohuashanensis[J]. Biodiversity Science, 2008, 16(6): 562−569. doi: 10.3321/j.issn:1005-0094.2008.06.006 [20] 李涛, 许丽, 陈鹏飞, 等. 北京西山地区花楸适应性研究[J]. 林业科技通讯, 2017(10): 77−79.Li T, Xu L, Chen P F, et al. Study on the adaptability of Sorbus pohuashanensis in xishan area of Beijing[J]. Forest Science and Technology, 2017(10): 77−79. [21] 张振英. 遮阴和施肥对黄山花楸幼苗生长及生理的影响[D]. 南京: 南京林业大学, 2012.Zhang Z Y. Effects of shading and fertilization on the growth and physiology of Huangshan Sorbus seedlings[D]. Nanjing: Nanjing Forestry University, 2012. [22] 刘迪. 花楸引种北京高平原和低山地区的耐热性研究[D]. 北京: 北京林业大学, 2020.Liu D. Study on the heat tolerance of Sorbus in the high plains and low mountain areas of Beijing[D]. Beijing: Beijing Forestry University, 2020. [23] 刘迪, 孙操稳, 王立宪, 等. 不同产地花楸无性系苗期田间耐热性评价研究[J]. 北京林业大学学报, 2020, 42(04): 21−31. doi: 10.12171/j.1000-1522.20190266Liu D, Sun C W, Wang L X, et al. Evaluation of field heat tolerance at seedling stage of Sorbus clone from different producing areas[J]. Journal of Beijing Forestry University, 2020, 42(04): 21−31. doi: 10.12171/j.1000-1522.20190266 [24] 苗胜越. 不同种源花楸越夏能力比较研究[D]. 保定: 河北农业大学, 2019.Miao S Y. Comparative study on the ability of Sorbus with different provenances[D]. Baoding: Hebei Agricultural University, 2019. [25] 闫铭. 花楸树种子育苗关键技术研究[D]. 晋中: 山西农业大学, 2019.Yan M. Research on the key technology of sorbus tree seedling cultivation[D]. Jinzhong: Shanxi Agricultural University, 2019. [26] 李建军. 几种花楸属植物繁殖技术研究进展[J]. 北方果树, 2017(1): 1−3.Li J J. Research progress on reproduction technology of several Sorbus plants[J]. Northern Fruit Tree, 2017(1): 1−3. [27] 王雅婧. 山西省野生花楸的园林应用价值评价与繁殖方法研究[D]. 晋中: 山西农业大学, 2015.Wang Y J. Research on the garden application value evaluation and propagation methods of wild Sorbus in Shanxi province[D]. Jinzhong: Shanxi Agricultural University, 2015. [28] 肖乾坤, 张川红, 郑勇奇. 影响花楸树嫩枝扦插成活率关键因素分析[J]. 河北农业大学学报, 2010, 33(3): 67−71. doi: 10.3969/j.issn.1000-1573.2010.03.015Xiao Q K, Zhang C H, Zheng Y Q. Analysis of key factors affecting the survival rate of tenderwood cuttings of Sorbus pohuashanensis[J]. Journal of Hebei Agricultural University, 2010, 33(3): 67−71. doi: 10.3969/j.issn.1000-1573.2010.03.015 [29] 赵洋, 陈云, 张晓晨, 等. 遮阴对少叶花楸幼苗光合和叶绿素荧光参数的影响[J]. 东北林业大学学报, 2019, 47(1): 30−34. doi: 10.3969/j.issn.1000-5382.2019.01.006Zhao Y, Chen Y, Zhang X C, et al. Effects of shading on the photosynthesis and chlorophyll fluorescence parameters of Sorbus pohuashanensis seedlings[J]. Journal of Northeast Forestry University, 2019, 47(1): 30−34. doi: 10.3969/j.issn.1000-5382.2019.01.006 [30] 彭松, 郑勇奇, 马淼, 等. 高温胁迫下花楸树幼苗的生理响应[J]. 林业科学研究, 2011, 24(5): 602−608.Peng S, Zheng Y Q, M, et al. Physiological response of Sorbus seedlings under high temperature stress[J]. Forestry Science Research, 2011, 24(5): 602−608. [31] Zhang Z, Pei X, Zhang R, et al. Molecular characterization and expression analysis of small heat shock protein 17.3 gene from Sorbus pohuashanensis (Hance) Hedl. in response to abiotic stress[J]. Molecular biology reports, 2020, 47(12): 1−11. [32] 张泽, 裴鑫, 鲁仪增, 等. 花楸树小热激蛋白23.8基因(SpHSP23.8)克隆与表达分析[J]. 植物资源与环境学报, 2020, 29(5): 9−20. doi: 10.3969/j.issn.1674-7895.2020.05.02Zhang Z, Pei X, Lu Y Z, et al. Cloning and expression analysis of Sorbus pohuashanensis small heat shock protein 23.8 Gene (SpHSP23.8)[J]. Journal of Plant Resources and Environment, 2020, 29(5): 9−20. doi: 10.3969/j.issn.1674-7895.2020.05.02 [33] 刘聪聪, 张泽, 关雪莲, 等. 花楸树热激蛋白70基因的克隆及表达分析[J]. 分子植物育种, 2019, 17(19): 6276−6286.Liu C C, Zhang Z, Guan X L, et al. Cloning and expression analysis of Sorbus pohuashanensis heat shock protein 70 gene[J]. Molecular Plant Breeding, 2019, 17(19): 6276−6286. [34] Pei X, Zhang Y, Zhu L Y, et al. Physiological and transcriptomic analyses characterized high temperature stress response mechanisms in Sorbus pohuashanensis[J]. Scientific Reports, 2021, 11: 10117. doi: 10.1038/s41598-021-89418-7 [35] 吴丹丹. SlNAC8提高转基因拟南芥抗逆性及机制分析[D]. 大连: 辽宁师范大学, 2018.Wu D D. SlNAC8 improves stress resistance of transgenic Arabidopsis and its mechanism analysis[D]. Dalian: Liaoning Normal University, 2018. [36] Sarkar N K, Kundnani P, Grover A. Functional analysis of Hsp70 superfamily proteins of rice (Oryza sativa)[J]. Cell Stress & Chaperones, 2013, 18(4): 427. [37] Ingolia T D, Craig E A. Drosophila gene related to the major heat shock-induced gene is transcribed at normal temperatures and not induced by heat shock[J]. PNAS, 1982, 79(2): 525−529. doi: 10.1073/pnas.79.2.525 [38] 屠小菊. 拟南芥DBB1α基因热响应的分子机理及原核表达[D]. 长沙: 湖南大学, 2010.Tu X J. Molecular mechanism and prokaryotic expression of Arabidopsis DBB1α gene thermal response[D]. Changsha: Hunan University, 2010. [39] 徐红云, 张明意. GRAS转录因子AtSCL4负调控拟南芥应答渗透胁迫[J]. 生物技术通报, 2022, 38(6): 129−135.Xu H Y, Zhang M Y. AtSCL4, an Arabidopsis thaliana GRAS transcription factor, negatively modulates plants in response to osmotic stress[J]. Biotechnology Bulletin, 2022, 38(6): 129−135. [40] Liu H C, Liu H T, Charng Y Y. The role of class A1 heat shock factors (HSFA1s) in response to heat and other stresses in Arabidopsis[J]. Plant, Cell & Environment, 2011, 34(5): 738−751. [41] Ikeda M, Mitsuda N, Ohme-Takagi M. Arabidopsis HsfB1 and HsfB2b act as repressors of the expression of heat-inducible Hsfs but positively regulate the acquired thermotolerance[J]. Plant Physiology, 2011, 157(3): 1243−1254. doi: 10.1104/pp.111.179036 [42] Tiwari L D, Khungar L, Grover A. AtHsc70-1 negatively regulates the basal heat tolerance in Arabidopsis thaliana through affecting the activity of HsfAs and Hsp101[J]. The Plant Journal:for cell and molecular biology, 2020, 103(6): 2069−2083. doi: 10.1111/tpj.14883 [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. -
点击查看大图
计量
- 文章访问数: 38
- HTML全文浏览量: 4
- PDF下载量: 4
- 被引次数: 0
