Expression and function analysis of histidine kinase gene PaHK3a of poplar ‘84K’
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.
表 1 qRT-PCR引物序列
Table 1. Primer sequences of qRT-PCR
Primer sequence (5′→3′)
PaHK3a-q-F CTCAGTTTCTTGCTACAGTTTCCC 243 PaHK3a-q-R ACATCATCCATTATTGCCCTCA Actin-F AAACTGTAATGGTCCTCCCTCCG 193 Actin-R GCATCATCACAATCACTCTCCGA
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
MDA 0.901 0.994** SOD 0.791 0.734 POD 0.954* 0.690
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