Expression patterns and salt tolerance analysis of BpPAT1 gene in Betula platyphylla
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摘要:
目的 GRAS家族是植物特有的具有高度保守羧基末端的转录因子家族，已有研究表明GRAS转录因子是植物胁迫反应中关键的转录调节因子之一。本研究拟对白桦中GRAS转录因子基因BpPAT1基因是否具有耐盐能力进行分析，为阐明白桦GRAS转录因子响应盐胁迫的分子调控机制奠定基础，进一步丰富木本植物GRAS转录因子响应逆境胁迫分子机制的研究。 方法 从盐胁迫白桦转录组数据中筛选并获得了1条GRAS转录因子基因，将其命名为BpPAT1。利用蛋白多序列比对及系统进化树来分析BpPAT1与其他GRAS家族蛋白的亲缘关系。利用实时荧光定量PCR（qRT-PCR）技术分析盐胁迫及非胁迫条件下白桦根、茎和叶组织中BpPAT1的表达模式，初步鉴定其是否响应盐胁迫。为了进一步分析BpPAT1的抗逆功能，构建其植物过表达载体（pROKII-BpPAT1）与抑制表达载体（pFGC5941-BpPAT1），利用农杆菌介导的高效瞬时遗传转化体系，获得BpPAT1基因瞬时过表达、抑制表达及对照白桦植株。在盐胁迫下分别对BpPAT1瞬时表达及对照植株的耐盐相关生理指标进行测定，鉴定BpPAT1是否能调控白桦的耐盐能力。 结果 多序列比对及系统进化树分析结果表明BpPAT1蛋白具有GRAS家族的序列特征，且与拟南芥中AtPAT1蛋白的亲缘关系较近。qRT-PCR结果表明：在盐胁迫6 h后，BpPAT1在白桦植株中的表达量显著上升（P < 0.05），说明该基因能响应盐胁迫。抗逆生理指标的测定结果表明：在白桦中过表达BpPAT1能够使过氧化物酶（POD）及超氧化物歧化酶（SOD）活性显著增强（P < 0.05），同时增加了白桦组织中的脯氨酸含量，降低了电解质渗透率及丙二醛含量。 结论 白桦BpPAT1基因能响应盐胁迫，过表达BpPAT1显著增加了白桦POD、SOD酶活性和脯氨酸含量，降低了电解质渗透率及丙二醛含量，进而提高了ROS清除能力，有效增强了白桦的耐盐能力。 Abstract:Objective GRAS family is a plant-specific transcription factor family, characterized by a highly conserved carboxyl terminus domain. Previous studies have shown that GRAS transcription factor is one of the key transcriptional regulators in plant stress response. The purpose of this study is to analyze the salt tolerance of GRAS transcription factor gene BpPAT1 gene in Betula platyphylla, so as to lay a foundation for elucidating the molecular regulation mechanism of GRAS transcription factor in response to salt stress. Our work enriched the research on the molecular mechanism of the GRAS transcription factors of woody plant in response to stress. Method In this study, one GRAS transcription factor gene was screened from the transcriptome data of B. platyphylla under salt stress and named as BpPAT1. Multiple sequence alignment and phylogenetic tree were used to analyze the genetic relationship between BpPAT1 and other organism’s GRAS family genes. Real-time fluorescence quantitative PCR (qRT-PCR) method was used to analyze the expression pattern of BpPAT1 in root, stem and leaf tissues of B. platyphylla under salt stress and normal condition, to identify whether it responded to salt stress or not. In order to further analyze the stress tolerance function of BpPAT1, plant overexpression vector (pROKII-BpPAT1) and inhibitory expression vector (pFGC5941-BpPAT1) were constructed. Transient overexpression and inhibitory expression of BpPAT1 gene and control B. platyphylla plants were obtained by Agrobacterium tumefaciens-mediated transient genetic transformation system. The physiological indexes related to salt tolerance were measured to identify whether the BpPAT1 was associated with salt tolerance in transient expression of BpPAT1 and control plants under salt stress. Result The results of multiple sequence alignment and phylogenetic tree analysis showed that BpPAT1 protein had the sequence characteristics of GRAS family and was closely related to AtPAT1 protein in A. thaliana. The result level of qRT-PCR showed that the expression of BpPAT increased significantly in B. platyphylla plants after 6 hours of salt stress, indicating that BpPAT1 could respond to salt stress signal. The measurement results of the physiological indexes of stress resistance showed that the overexpression of BpPAT1 in B. platyphylla could significantly increase the activity of peroxidase (POD) and superoxide dismutase (SOD), increased the content of proline, and decreased electrolyte leakage and malondialdehyde (MDA) content. Conclusion The BpPAT1 gene can respond to salt stress, overexpression of BpPAT1 significantly enhances POD, SOD enzyme activities and proline content, decreases electrolyte leakage and MDA content under salt stress, thus improves ROS scavenging ability and salt tolerance of B. platyphylla. -
Key words:
- Betula platyphylla /
- GRAS transcription factor /
- BpPAT1 /
- gene expression /
- salt stress response
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图 1 白桦BpPAT1蛋白的多序列比对分析(A)及系统进化树分析(B)
AtPAT1. 拟南芥(NP_001332482.1)；QsGRAS. 欧洲栓皮栎(XP_023916980.1)；MrGRAS. 杨梅(KAB1208676.1)；JrGRAS. 胡桃(XP_018849898.1)；VvGRAS. 葡萄(XP_002272334.1)；JcGRAS20. 麻风树(XP_012081428.1)；CfGRAS. 土瓶草(GAV74587.1)；DlGRAS54. 龙眼(AGE44291.1)；TcGRAS. 可可(EOX93442.1)。AtPAT1, Arabidopsis thaliana (NP_001332482.1); QsGRAS, Quercus suber (XP_023916980.1); MrGRAS, Morella rubra (KAB1208676.1); JrGRAS, Juglans regia (XP_018849898.1)；VvGRAS, Vitis vinifera (XP_002272334.1); JcGRAS20, Jatropha curcas (XP_012081428.1); CfGRAS, Cephalotus follicularis (GAV74587.1); DlGRAS54, Dimocarpus longan (AGE44291.1); TcGRAS, Theobroma cacao (EOX93442.1).
Figure 1. Multiple sequence alignment analysis of Betula platyphylla BpPAT1 protein (A) and phylogenetic tree analysis (B)
图 2 盐胁迫条件下BpPAT1基因的表达模式
Figure 2. Expression pattern of BpPAT1 in B. platyphylla under salt stress
图 3 盐胁迫下BpPAT1基因在瞬时表达白桦植株中的表达情况
A. 盐胁迫下不同转化时间对照植株及瞬时过表达植株BpPAT1基因的表达水平；B. 正常条件下与盐胁迫后对照植株及瞬时转化植株BpPAT1基因的表达水平；Control. 对照植株；OE. 瞬时过表达植株；IE. 瞬时抑制表达植株；*. 显著性差异(P < 0.05)。下同。A, expression levels of BpPAT1 gene in control plants and transient overexpressed plants at different transformation time under salt stress; B, expression level of BpPAT1 gene in control plants and transient transformed plants under normal conditions and salt stress. Control, control plant; OE, transient overexpression plant; IE, transient inhibitory expression plant. Asterisks indicate significant difference (P < 0.05). The same below.
Figure 3. Expression of BpPAT1 gene in transient expression plants of B. platyphylla under salt stress
图 4 正常条件及盐胁迫后瞬时表达OE、IE及Control白桦植株中ROS水平(B、D)及POD(A)、SOD(C)活性分析
Figure 4. Analysis of ROS level (B, D) and activities of POD (A) and SOD (C) in OE, IE and control B. platyphylla plants under normal condition and salt stress
图 5 正常条件及盐胁迫下OE、IE及Control白桦植株中电解质渗透率(A)及MDA含量(B)分析
Figure 5. Electrolyte leakage (A) and MDA content (B) analysis in OE, IE and control B. platyphylla plants under normal condition and salt stress
图 6 正常条件及盐胁迫下OE、IE及Control白桦植株中脯氨酸含量分析
Figure 6. Proline content analysis in OE，IE and control B. platyphylla plants under normal condition and salt stress
表 1 引物序列
Table 1. Primers used in this study
用途 Application 引物名称 Primer name 引物序列(5′—3′) Primer sequence (5′−3′) 实时荧光定量 PCR Quantitative real-time PCR q-BpPAT1-F TACTGCTGCATTCTATCCAC q-BpPAT1-R ACTTACCAAGAAGCTCATG q-BpPAT1-OE-F CCCCACATCCGCATAACA q-BpPAT1-OE-R CCCAGGTCGAATCCCAAG q-BpPAT1-IE-F ACGAACGGTGTTGCACTT q-BpPAT1-IE-R GAGCCATAGGTATTGTCAGG Actin-F TGAGAAGAGCTATGAGTTGC Actin-R GTAGATCCACCACTAAGCAC Tubulin-F TCAACCGCCTTGTCTCTCAGG Tubulin-R TGGCTCGAATGCACTGTTGG 基因克隆 Gene cloning BpPAT1-F GCTCTAGAATGTCCAACGGATTGTACTATC BpPAT1-R GGGTACCTCACTTCCATGCACAAGCAG 载体构建 Vector verification pROKⅡ-F AGACGTTCCAACCACGTCTT pROKⅡ-R CCAGTGAATTCCCGATCTAG pFGC5941-cisF CGCTCGAGTATAAGAGCT pFGC5941-cisR ACCTTCCCACAATTCGTCGG pFGC5941-antiF GCATGCTATGCATTCAAT pFGC5941-antiR CGTGCACAACAGAATTGAAAGC pFGC5941-BpPAT1-cisF CCCATGGCAGCTATGCTACAATGATAG pFGC5941-BpPAT1-cisR TTGGCGCGCCTCCACATATAGAAGAGCCAT pFGC5941-BpPAT1-antiF CTCTAGACAGCTATGCTACAATGATAG pFGC5941-BpPAT1-antiR CGGATCCTCCACATATAGAAGAGCCAT 
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