Expression and function analysis of histidine kinase gene PaHK3a of poplar ‘84K’
-
摘要:
目的 干旱、高盐等逆境胁迫严重影响了植物的生长发育。研究表明，组氨酸激酶在植物逆境响应中起重要作用。本研究对银腺杨‘84K’组氨酸激酶基因PaHK3a在不同组织的表达模式分析，检测了其对生长素、细胞分裂素等植物激素处理下及人工干旱、盐碱等非生物胁迫下的表达，结合干旱、盐碱条件下丙二醛（MDA）及保护酶活性等生化指标，对该基因的功能进行了初步鉴定，为杨树抗逆分子育种研究奠定基础。 方法 以‘84K’杨无菌苗为材料，通过实时定量PCR（qRT-PCR）技术分析PaHK3a基因在不同组织的表达模式。对‘84K’杨无菌苗进行浓度为10 mmol/L植物激素处理（ABA、6-BA、IBA、GA3及水杨酸（SA））及非生物胁迫处理（42 ℃高温、0 ℃低温、200 mmol/L NaCl和5% PEG6000），采用qRT-PCR技术分析PaHK3a基因对不同植物激素及非生物胁迫的表达响应；进一步对温室‘84K’杨进行自然干旱处理（6、8、10 d）、200 mmol/L NaCl（2、4、6 d）处理，测定不同胁迫时间点叶片PaHK3a基因的表达，以及超氧化物歧化酶（SOD）、过氧化物酶（POD）活性及MDA含量，并分析PaHK3a基因表达与生理指标的相关性，初步鉴定杨树PaHK3a基因的功能。 结果 qRT-PCR结果显示，PaHK3a基因在叶片中表达量最高，根部中等，茎段最低。与正常条件下相比，在高温、低温、NaCl及PEG模拟干旱处理时，PaHK3a基因表达量与对照相比明显增高，分别为对照的2.63、1.49、1.54、1.58倍。用IBA诱导处理时，基因表达量与对照相比差异不大，而在6-BA、ABA、GA3及SA处理时，基因表达量与对照相比均呈现显著下调。在温室干旱、盐碱胁迫处理过程中，PaHK3a基因表达均显著高于对照，呈现先上升后下降的表达模式，MDA含量也呈现类似的趋势，而SOD活性则随处理时间的延长而持续升高，POD活性在干旱胁迫时先上升后下降，而高盐胁迫时呈上升趋势。生理指标与PaHK3a基因表达量相关系分析发现，在干旱和盐胁迫下，PaHK3a基因表达量与叶片MDA含量、SOD活性和POD活性均呈正相关。 结论 PaHK3a基因在‘84K’杨根茎叶中均有表达，且叶中表达量最高；PaHK3a基因表达受细胞分裂素6-BA、GA3及ABA及SA等植物激素的负调控，同时，受温度胁迫、盐胁迫、水分胁迫等非生物胁迫正调控；温室人工干旱盐碱胁迫过程中，PaHK3a基因表达量升高，且与叶片MDA含量、SOD活性、POD活性均具有正相关性。研究结果初步显示，杨树PaHK3a基因参与杨树植物激素激素信号响应，并在抗逆境胁迫过程中发挥重要调控作用。 Abstract:Objective Abiotic stresses such as drought and salt seriously affect the growth and development of plants. Previous studies showed that histidine kinase played an important role during the process of plants responsing to abiotic stresses. In this study, we analyzed the expression of histidine kinase gene (PaHK3a) in roots, stems and leaves of poplar ‘84K’ (Poplus alba × P. glandulosa ‘84K’). And the expression of PaHK3a in leaves of in vitro ‘84K’ plants under various plant hormones and abiotic stresses was also detected. Together with the malondialdehyde (MDA) contents, superoxide dismutase (SOD) and peroxidase (POD) activities in leaves of poplar ‘84K’ in green house, the function of PaHK3a was preliminary proposed. The results in this study laid a foundation for molecular breeding of poplar for resistance. Method Using in vitro poplar ‘84K’ as materials, the expression of PaHK3a gene in different organs, under plant hormone treatments (10 mmol/L ABA, 10 mmol/L 6-BA, 10 mmol/L IBA, 10 mmol/L GA3 and 10 mmol/L SA) and various abiotic stress conditions (42 ℃, 0 ℃, 200 mmol/L NaCl, and 5% PEG6000), was analyzed by qRT-PCR. Meanwhile, the expression of PaHK3a in leaves of green house grown poplar ‘84K’ plants under drought (6, 8, 10 d) and salt stress (2, 4, 6 d) was also detected, and the MDA contents, SOD and POD activities were measured, and the correlation between expression of PaHK3a gene and physiological indicators was analyzed to determine the function of PaHK3a preliminarily. Result The expression of PaHK3a gene was highest in leaves, medium in roots and lowest in stems. The qRT-PCR results showed that the transcriptional levels of PaHK3a gene were about 2.63, 1.49, 1.54, 1.58 times of control, respectively under 42 ℃, 0 ℃, 200 mmol/L NaCl, and 5% PEG treatments. Under IBA treatment, the transcripts of PaHK3a were not significantly different from control. The expression of PaHK3a was down-regulated under the treatments of 6-BA, ABA, GA3 and SA, respectively. At different stress times of drought and salt treatment, the PaHK3a gene in leaves of greenhouse grown ‘84K’ plants increased significantly, with the style of increased first and then decreased; and the MDA content in leaves also had the similar style. SOD and POD activity were measured in drought and high salt, MDA content increased first and then decreased; SOD activity in leaves increased constantly during drought and salt stresses, and POD activity first increased and then decreased under drought stress, while increased constantly during salt stress. The correlation analysis between physiological indexes and PaHK3a gene expression found that under drought and salt stress, the expression of PaHK3a gene was positively correlated with MDA content, SOD activity and POD activity in leaves. Conclusion The PaHK3a was expressed in roots, stems and leaves of poplar ‘84K’, with highest expression in leaves. The expression of PaHK3a was down-regulated by exogenous cytokinin (6-BA, GA3) and stress related plant hormones (ABA, SA), and up-regulated by temperature, salt and drought stresses. During the process of drought and salt stresses, the expression of PaHK3a increased significantly, with the increase of MDA content, SOD activity and POD activity, and it was positively correlated with MDA content, SOD activity and POD activity in leaves. Our results indicate that PaHK3a is involved in the response of poplar to plant hormones and plays an important role in poplar response to abiotic stresses. -
Key words:
- P. alba × P. glandulosa ‘84K’ /
- histidine kinase /
- gene expression /
- abiotic stress
-
图 1 PaHK3a基因在‘84K’杨根、茎和叶中的表达情况
*表示差异显著，P < 0.05；**表示差异极显著，P < 0.01。下同。* means significant difference, P < 0.05; ** means very significant difference, P < 0.01. The same below.
Figure 1. Expression of PaHK3a in roots, stems and leaves of ‘84K’ poplar
图 2 非生物胁迫处理下‘84K’杨叶片PaHK3a基因的表达差异
CK. 对照；HT. 42 ℃高温处理；LT. 0 ℃低温处理。下同。CK, control; HT, 42 ℃ high temperature treatment; LT, 0 ℃ low temperature treatment. The same below.
Figure 2. Expression differences of PaHK3a in ‘84K’leaves under abiotic stresses
图 3 不同植物激素处理下‘84K’杨叶片PaHK3b基因的表达差异
Figure 3. Expression differences of PaHK3a in ‘84K’ leaves under several plant hormone treatments
图 4 ‘84K’杨温室干旱（A）及盐胁迫（B）下叶片PaHK3a基因表达量
Figure 4. Expression of PaHK3a in leaves of ‘84K’ under drought (A) and salt (B) stresses in greenhouse
图 5 ‘84K’杨温室干旱（A）及盐（B）胁迫下叶片MDA的含量
Figure 5. MDA contents in leaves of ‘84K’ under drought (A) and salt (B) stresses in greenhouse
图 6 ‘84K’杨温室干旱（A）及盐胁迫（B）下叶片SOD活性
Figure 6. SOD activity in leaves of ‘84K’ under drought (A) and salt (B) stresses in greenhouse
图 7 ‘84K’杨温室干旱（A）及盐胁迫（B）下叶片POD活性
Figure 7. POD activity in leaves of ‘84K’ under drought (A) and salt (B) stresses in greenhouse
表 1 qRT-PCR引物序列
Table 1. Primer sequences of qRT-PCR
引物名称
Primer name引物序列(5′→3′)
Primer sequence (5′→3′)产物长度
Product length/bpPaHK3a-q-F CTCAGTTTCTTGCTACAGTTTCCC 243 PaHK3a-q-R ACATCATCCATTATTGCCCTCA Actin-F AAACTGTAATGGTCCTCCCTCCG 193 Actin-R GCATCATCACAATCACTCTCCGA 
下载: 导出CSV
表 2 干旱、盐胁迫下PaHK3a基因表达与生理指标相关性分析
Table 2. Correlation analysis between expression of PaHK3a and physiological indexes under drought and salt stresses
生理指标
Physiological index干旱胁迫
Drought stress盐胁迫
Salt stressMDA 0.901 0.994** SOD 0.791 0.734 POD 0.954* 0.690
下载: 导出CSV
-
[1] Muller B, Sheen J. Arabidopsis cytokinin signaling pathway [J/OL]. Science Signaling STKE, 2007, 2007(407): cm5 [2020−02−13]. https://stke.sciencemag.org/content/2007/407/cm5. [2] Werner T, Schmulling T. Cytokinin action in plant development[J]. Current Opinion in Plant Biology, 2009, 12(5): 527−538. [3] Tran L S, Urao T, Qin F, et al. Functional analysis of AHK1/ATHK1 and cytokinin receptor histidine kinases in response to abscisic acid, drought, and salt stress in Arabidopsis[J]. PNAS, 2007, 104(51): 20623−20628. [4] Jeon J, Kim N Y, Kim S, et al. A subset of cytokinin two-component signaling system plays a role in cold temperature stress response in Arabidopsis[J]. Journal of Biological Chemistry, 2010, 285: 23371−23386. [5] Sheen J. Phosphorelay and transcription control in cytokinin signal transduction[J]. Science, 2002, 296: 1650−1652. [6] Heyl A, Schmülling T. Cytokinin signal perception and transduction[J]. Current Opinion in Plant Biology, 2003, 6(5): 480−488. [7] Schaller F, Zerbe P, Reinbothe S, et al. The allene oxide cyclase family of Arabidopsis thaliana: localization and cyclization[J]. FEBS Journal, 2008, 275(10): 2428−2441. [8] Hwang I, Chen H C, Sheen J, et al. Two-component signal transduction pathways in Arabidopsis[J]. Plant Physiol, 2002, 129(5): 500−515. [9] Maruyama-Nakashita A, Nakamura Y, Yamaya T, et al. A novel regulatory pathway of sulfate uptake in Arabidopsis roots: implication of CRE1/WOL/AHK4 mediated cytokinin-dependent regulation[J]. Plant Journal, 2004, 38(5): 779−789. [10] Chefdor F, Héricourt F, Koudounas K, et al. Highlighting type ARRs as potential regulators of the dkHK1 multi-step phosphorelay pathway in Populus[J]. Plant Science, 2018, 277: 68−78. [11] Kim H J, Ryu H, Hong S H, et al. Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in Arabidopsis[J]. Proc Natl Acad Sci USA, 2006, 103(3): 814−819. [12] Riefler M, Novsk O, Strnad M, et al. Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism[J]. Plant Cell, 2006, 18: 40−54. [13] Dello I R, Linhares F S, Scacchi E, et al. Cytokinins determine Arabidopsis root meristem size by controlling cell differentiation[J]. Current Biology, 2007, 17(8): 678−682. [14] Higuchi M, Pischke M S , Mhnen A P , et al. In planta functions of the Arabidopsis cytokinin receptor family[J]. PNAS, 2004, 101(23): 8821−8826. [15] Bartrina I, Jensen H, Novák O, et al. Gain-of-function mutants of the cytokinin receptors AHK2 and AHK3 regulate plant organ size, flowering time and plant longevity[J]. Plant Physiology, 2007, 173(3): 1783−1797. [16] Kang N Y, Cho C, Kim N Y, et al. Cytokinin receptor-dependent and receptor-independent pathways in the dehydration response of Arabidopsis thaliana[J]. Journal of Plant Physiology, 2012, 169(14): 1382−1391. [17] Jin J, Nan Y K, Sunmi K, et al. A subset of cytokinin two-component signaling system plays a role in cold temperature stress response in Arabidopsis[J]. Journal of Biological Chemistry, 2010, 285(30): 23371−23386. [18] Cortleven A, Nitschke S, Klaumünzer M, et al. A novel protective function for cytokinin in the light stress response is mediated by the Arabidopsis histidine kinase2 and Arabidopsis histidine kinase3 receptors[J]. Plant Physiology, 2014, 164(3): 1470−1483. [19] Nieminen K, Immanen J, Laxell M, et al. Cytokinin signaling regulates cambial development in poplar[J]. Proceedings of the National Academy of Sciences, 2008, 105(50): 20032−20037. [20] Sun J, Niu Q W, Tarkowski P, et al. The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis[J]. Plant Physiology, 2003, 131(1): 167−176. [21] Verslues P E. ABA and cytokinins: challenge and opportunity for plant stress research[J]. Plant Molecular Biology, 2016, 91(6): 629−640. [22] Tran L S, Shinozaki K, Yamaguchi-Shinozaki K, et al. Role of cytokinin responsive two component system in ABA and osmotic stress signaling[J]. Plant Signal & Behavior, 2010, 5(2): 148−150. [23] Ramsong N, Praveen S, Ratna K, et al. Histidine kinases in plants: cross talk between hormone and stress responses[J]. Plant Signaling & Behavio, 2012, 7(10): 1230−1237. [24] Wang B, Guo B, Xie X, et al. A novel histidine kinase gene, ZmHK9, mediate drought tolerance through the regulation of stomatal development in Arabidopsis[J]. Gene, 2012, 501(2): 171−179. [25] Vernooij B, Friedrich L, Weymann K, et al. A central role of salicylic acid in plant disease resistance[J]. Science, 1994, 266: 1247−1250. [26] Yusuf M, Hasan S A, Ali B, et al. Effect of salicylic acid on salinity-induced changes in Brassica juncea[J]. Journal of Integrative Plant Biology, 2008, 50(9): 1096−1102. [27] Brito G, Costa A, Fonseca H M A C, et al. Response of Olea europaea ssp. maderensis in vitro shoots exposed to osmotie stress[J]. Scientia Horticulturae, 2003, 97(S3−S4): 411−417. [28] 张磊, 谢锦忠, 张玮, 等. 模拟干旱环境下伐桩注水对毛竹生理特性的影响[J]. 林业科学研究, 2017, 30(1):145−153.Zhang L, Xie J Z, Zhang W, et al. Effects of water storage in bamboo stumps on physiological characteristicsof phyllostachys edulis under simulated drought environment[J]. Forest Research, 2017, 30(1): 145−153. [29] 党晓宏, 高永, 蒙仲举, 等. 3种滨藜属植物幼苗叶片对NaCl胁迫的生理响应[J]. 北京林业大学学报, 2016, 38(10):38−49.Dang X H, Gao Y, Meng Z J, et al. Leaf physiological characteristics of seedlings of three Atriplex species under NaCl stress[J]. Journal of Beijing Forestry University, 2016, 38(10): 38−49. [30] Sofo A, Dichio B, Xiloyannis C, et al. Effects of different irradiance levels on some antioxidant enzymes and on malondialdehyde content during rewatering in olive tree[J]. Plant Science, 2004, 166(2): 293−302. [31] Kong J, Dong Y, Xu L, et al. Role of exogenous nitric oxide in alleviating iron deficiency-induced peanut chlorosis on calcareous soil[J]. Journal of Plant Interactions, 2014, 9(1): 450−459. -
点击查看大图
图(7) / 表(2)
计量
- 文章访问数: 829
- HTML全文浏览量: 320
- PDF下载量: 45
- 被引次数: 0
