| Horticulture |
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| Cloning of PsDHN1 gene of Paeonia suffruticosa and waterlogging tolerance analysis of transgenic Arabidopsis with PsDHN1 gene |
Huichun LIU( ),Jiaqiang ZHANG,Guangying MA,Jianghua ZHOU,Wenting XU,Kaiyuan ZHU() |
| Zhejiang Institute of Landscape Plants and Flowers, Hangzhou 311251, China |
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Abstract Based on the previous studies, an intermediate sequence of dehydrin gene PsDHN1 was obtained from the leaves of Paeonia suffruticosa and the full-length sequence of PsDHN1 gene was cloned by the rapid-amplification of cDNA ends (RACE) method. In order to explore the characteristics of PsDHN1 and its function in response to waterlogging stress, bioinformatic analysis and transformation into Arabidopsis for functional verification were performed. The results of bioinformatic analysis showed that the full length cDNA of PsDHN1 was 864 bp, containing an open reading frame (ORF) of 471 bp, and the lengths of 5′ and 3′ noncoding regions were 104 bp and 289 bp, respectively. The protein encoded by PsDHN1 gene contained 156 amino acids with the molecular mass of 16.39 kDa, theoretical isoelectric point of 8.87, hydrophilic index of －1.274, belonging to hydrophilic protein, and instability index of 44.05, belonging to unstable protein. The PsDHN1 protein contained three α-helixes and one β-flod, and two Y fragments, one S fragment and two K fragments that belonged to the typical Y2SK2 dehydratin. In addition, the PsDHN1 protein of P. suffruticosa had some homologies with other nine species, among which the similarities between P. suffruticosa and Momordica charantia, Jatropha curcas, Juglans regia were 58%, 54% and 53%, respectively. Subcellular localization results showed that PsDHN1 was mainly localized in the nucleus and cell membrane. The results of waterlogging tolerance test showed that the phenotype of transgenic Arabidopsis plants with PsDHN1 gene had better waterlogging resistance and growth recovery than the wild-type plants. Moreover, the physiological indexes that related to waterlogging tolerance such as sucrose synthase, pyruvate decarboxylase, α-amylase activities and soluble protein content reflected that transgenic Arabidopsis had higher waterlogging tolerance than wild-type plants. The results provide a theoretical basis for mining waterlogging tolerance genes and further research on the molecular mechanisms of P. suffruticosa.
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Received: 03 September 2020
Published: 25 June 2021
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Corresponding Authors:
Kaiyuan ZHU
E-mail: lhuichun@163.com;52011648@qq.com
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牡丹PsDHN1基因克隆及转基因拟南芥的耐涝性分析
在前期研究的基础上，从牡丹叶片中获得了脱水素基因PsDHN1的中间序列，利用cDNA末端快速扩增（rapid-amplification of cDNA ends, RACE）技术克隆获得了该基因的全长序列。通过生物信息学分析及转化拟南芥进行功能验证，探讨牡丹脱水素基因PsDHN1的特性及其响应涝害胁迫的功能。生物信息学分析结果表明：PsDHN1基因全长cDNA为864 bp，包括471 bp的开放阅读框，104 bp的5′非编码区和289 bp的3′非编码区。PsDHN1蛋白含有156个氨基酸，其分子质量为16.39 kDa，理论等电点为8.87，亲水性指数为－1.274，为亲水性蛋白，不稳定性指数为44.05，为不稳定性蛋白。PsDHN1蛋白包含3个α-螺旋和1个β-折叠，以及2个Y片段、1个S片段和2个K片段，属于典型的Y2SK2型脱水素。牡丹的PsDHN1蛋白与其他9个物种有一定的同源性，其中与苦瓜、麻风树和胡桃DHN1蛋白的相似度分别达到了58%、54%和53%。亚细胞定位结果表明，PsDHN1主要定位于细胞核和细胞膜上。耐涝性试验结果显示：从表型上看，转PsDHN1基因的拟南芥植株耐涝能力及恢复生长的情况均优于野生型植株；涝害相关的酶如蔗糖合成酶、丙酮酸脱羧酶、α-淀粉酶活性及可溶性蛋白含量均为转基因拟南芥总体高于野生型植株。该研究结果为牡丹耐涝基因的挖掘和进一步研究牡丹耐涝的分子机制提供了理论依据。
关键词：
牡丹,
PsDHN1基因,
基因克隆,
耐涝性
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|
| [1] |
POSAS F, CHAMBERS J R, HEYMAN J A, et al. The transcriptional response of yeast to saline stress. Journal of Biological Chemistry, 2000,275(23):17249-17255. DOI:10.1074/jbc.M910016199
doi: 10
|
|
|
| [2] |
FUJITA M, FUJITA Y, NOUTOSHI Y, et al. Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Current Opinion in Plant Biology, 2006,9(4):436-442. DOI:10.1016/j.pbi.2006.05.014
doi: 10.1016/j.pbi.2006.05.014
|
|
|
| [3] |
SHINOZAKI K, YAMAGUCHI-SHINOZAKI K. Gene networks involved in drought stress response and tolerance. Journal of Experimental Botany, 2007,58(2):221-227. DOI:10.1093/jxb/erl164
doi: 10.1093/jxb/erl164
|
|
|
| [4] |
DURE L, CROUCH M, HARADA J, et al. Common amino acid sequence domains among the LEA proteins of higher plants. Plant Molecular Biology, 1989,12:475-486.
|
|
|
| [5] |
SAVEDRA L, SVESSON J, CARBALLO V, et al. A dehydrin gene in Physcomitrella patens is required for salt and osmotic stress tolerance. The Plant Journal, 2006,45(2):237-249. DOI:10.1111/j.1365-313X.2005.02603.x
doi: 10.1111/j.1365-313X.2005.02603.x
|
|
|
| [6] |
夏惠,林玲,高帆,等.植物脱水素对多种逆境的响应.干旱地区农业研究,2014,32(4):47-52. DOI:10.7606/j.issn.1000-7601.2014.04.009
XIA H, LIN L, GAO F, et al. Response of plant dehydrin to various stresses. Agricultural Research in the Arid Areas, 2014,32(4):47-52. (in Chinese with English abstract)
doi: 10.7606/j.issn.1000-7601.2014.04.009
|
|
|
| [7] |
HANIN M, BRINI F, EBEL C, et al. Plant dehydrins and stress tolerance: versatile proteins for complex mechanisms. Plant Signaling & Behavior, 2011,6(10):1503-1509. DOI:10.4161/psb.6.10.17088
doi: 10
|
|
|
| [8] |
HARA M, FUJINAGA M, KUBOI T. Radical scavenging activity and oxidative modification of citrus dehydrin. Plant Physiology and Biochemistry, 2004,42(7/8):657-662. DOI:10.1016/j.plaphy.2004.06.004
doi: 10
|
|
|
| [9] |
张宁,孙敏善,刘露露,等.小麦脱水素基因TaDHN-1的特征及其对非生物胁迫响应.中国农业科学,2013,46(4):849-858. DOI:10.3864/j.issn.0578-1752.2013.04.019
ZHANG N, SUN M S, LIU L L, et al. Characterization of a dehydrin gene TaDHN-1 and its response to abiotic stresses in wheat. Scientia Agricultura Sinica, 2013,46(4):849-858. (in Chinese with English abstract)
doi: 10.3864/j.issn.0578-1752.2013.04.019
|
|
|
| [10] |
JYOTHI-PRAKASH P A, MOHANTY B, WIJAYA E, et al. Identification of salt gland-associated genes and characteri-zation of a dehydrin from the salt secretor mangrove Avicennia officinalis. BMC Plant Biology, 2014,14:291. DOI:10.1186/s12870-014-0291-6
doi: 10.1186/s12870-014-0291-6
|
|
|
| [11] |
OCHOA-ALFARO A E, RODRíGUEZ-KESSLER M, PéREZ-MORALES M B, et al. Functional characterization of an acidic SK3 dehydrin isolated from an Opuntia streptacantha cDNA library. Planta, 2012,235:565-578. DOI:10.1007/s00425-011-1531-8
doi: 10.1007/s00425-011-1531-8
|
|
|
| [12] |
SUN J, NIE L Z, SUN G Q, et al. Cloning and characterization of dehydrin gene from Ammopiptanthus mongolicus. Molecular Biology Reports, 2013,40(3):2281-2291. DOI:10.1007/s11033-012-2291-7
doi: 10.1007/s11033-012-2291-7
|
|
|
| [13] |
PARK S Y, NOH K J, YOO J H, et al. Rapid upregulation of dehydrin3 and dehydrin4 in response to dehydration is a characteristic of drought tolerant genotype in barley. Journal of Plant Biology, 2006,49(6):455-462. DOI:10.1007/BF03031126
doi: 10.1007/BF03031126
|
|
|
| [14] |
HUNDERTMARK M, HINCHA D K. LEA (late embryo-genesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics, 2008,9(1):118. DOI:10.1186/1471-2164-9-118
doi: 10.1186/1471-2164-9-118
|
|
|
| [15] |
王俊娟,穆敏,王帅,等.棉花脱水素GhDHN1的克隆及其表达.中国农业科学,2016,49(15):2867-2878. DOI:10.3864/j.issn.0578-1752.2016.15.002
WANG J J, MU M, WANG S, et al. Molecular clone and expression of GhDHN1 gene in cotton (Gossypium hirsutum L.). Scientia Agricultura Sinica, 2016,49(15):2867-2878. (in Chinese with English abstract)
doi: 10.3864/j.issn.0578-1752.2016.15.002
|
|
|
| [16] |
ROYCHOUDHURY A, ROY C, SENGUPTA D N. Trans-genic tobacco plants overexpression the heterologous lea gene Rab16A from rice during high salt and water deficit display enhanced tolerance to salinity stress. Plant Cell Reports, 2007,26:1839-1859. DOI:10.1007/s00299-007-0371-2
doi: 10.1007/s00299-007-0371-2
|
|
|
| [17] |
MUNOZ-MAYOR A, PINEDA B, GARCIA-ABELLAN J O, et al. Overexpression of dehydrin tas14 gene improves the osmotic stress imposed by drought and salinity in tomato. Journal of Plant Physiology, 2012,169(5):459-468. DOI:10.1016/j.jplph.2011.11.018
doi: 10.1016/j.jplph.2011.11.018
|
|
|
| [18] |
KOVACS D, KALMAR E, TOROK Z, et al. Chaperone activity of ERD10 and ERD14, two disordered stress-related plant proteins. Plant Physiology, 2008,147:381-390. DOI:10.1104/pp.108.118208
doi: 10
|
|
|
| [19] |
PENG Y, REYES J L, WEI H, et al. RcDhn5, a cold acclimation responsive dehydrin from Rhododendron catawbiense rescues enzyme activity from dehydration effects in vitro and enhances freezing tolerance in RcDhn5-overexpression Arabidopsis plants. Physiologia Plantarum, 2008,134:583-597. DOI:10.1111/j.1399-3054.2008.01164.x
doi: 10.1111/j.1399-3054.2008.01164.x
|
|
|
| [20] |
FAN Z, WANG X. Isolation and characterization of a novel dehydrin gene from Capsella bursa-pastoris. Molecular Biology, 2006,40(1):43-50. DOI:10.1134/S0026893306010080
doi: 10.1134/S0026893306010080
|
|
|
| [21] |
ALSHEIKH M K, HEYEN B J, RANDALL S K. Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation. Journal of Biology Chemistry, 2003,278(42):40882-40889. DOI:10.1074/jbc.M307151200
doi: 10.1074/jbc.M307151200
|
|
|
| [22] |
史学英,田野,李核,等.小麦K2型脱水蛋白DHN14响应非生物胁迫的功能分析.西北农林科技大学学报(自然科学版),2019,47(5):23-41. DOI:10.13207/j.cnki.jnwafu.2019.05.004
SHI X Y, TIAN Y, LI H, et al. Functional analysis of K2-type wheat dehydrin DHN14 under abiotic stresses. Journal of Northwest A&F University (Natural Science Edition), 2019,47(5):23-41. (in Chinese with English abstract)
doi: 10.13207/j.cnki.jnwafu.2019.05.004
|
|
|
| [23] |
KOSOVá K, VíTáMVáS P, PRá?IL I T. Wheat and barley dehydrins under cold, drought, and salinity-what can LEA-Ⅱ protein tell us about plant stress response. Frontiers in Plant Science, 2014,5:343. DOI:10.3389/fpls.2014.00343
doi: 10.3389/fpls.2014.00343
|
|
|
| [24] |
ZHOU Y, HE P, XU Y P, et al. Overexpression of CsLEAⅡ, a Y3SK2-type dehydrin gene from cucumber (Cucumis sativus), enhances tolerance to heat and cold in Escherichia coli. AMB Expression, 2017,7(1):182. DOI:10.1186/s13568-017-0483-1
doi: 10.1186/s13568-017-0483-1
|
|
|
| [25] |
VASEVA I I, ANDERS I, FELLER U. Identification and expression of different dehydrin subclasses involved in the drought response of Trifolium repens. Journal of Plant Physiology, 2014,171(3/4):213-224. DOI:10.1016/j.jplph.2013.07.013
doi: 10.1016/j.jplph.2013.07.013
|
|
|
| [26] |
VLAD F, TURK B E, PEYNOT P, et al. A versatile strategy to define the phosphorylation preferences of plant protein kinases and screen for putative substrates. The Plant Journal, 2008,55:104-117. DOI:10.1111/j.1365-313X.2008.03488.x
doi: 10.1111/j.1365-313X.2008.03488.x
|
|
|
| [27] |
HALDER T, UPADHYAYA G, RAY S. YSK2 type dehydrin (SbDhn1) from Sorghum bicolor showed improved protection under high temperature and osmotic stress condition. Frontiers in Plant Science, 2017,8:918. DOI:10.3389/fpls.2017.00918
doi: 10.3389/fpls.2017.00918
|
|
|
| [28] |
CHEN Y, MA Y, GUO J, et al. Cloning and expression analysis of the dehydrin gene PIDHN1 in peony (Paeonia lactiflora). The Journal of Horticultural Science and Biotechnology, 2018,93(6):557-565. DOI:10.1080/14620316.2018.1431060
doi: 10.1080/14620316.2018.1431060
|
|
|
| [29] |
JACKSON M B, RAM P C. Physiological and molecular basis of susceptibility and tolerance of rice plants to complete submergence. Annals of Botany, 2003,91:227-241. DOI:10.1093/aob/mcf242
doi: 10.1093/aob/mcf242
|
|
|
| [30] |
刘慧春,张加强,周江华,等.牡丹PsGRP基因克隆及转基因拟南芥的耐涝性分析.植物生理学报,2021,57(2):373-384. DOI:10.13592/j.cnki.ppj.2020.0466
LIU H C, ZHANG J Q, ZHOU J H, et al. Cloning of PsGRP gene from Paeonia sufftruticosa and waterlogging tolerance analysis of transgenic Arabidopsis. Plant Physiology Journal, 2021,57(2):373-384. (in Chinese with English abstract)
doi: 10.13592/j.cnki.ppj.2020.0466
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