Cloning and expression analysis of BpSPL6 promoter from Betula platyphylla
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摘要:
目的 SPL是植物特有的转录因子，参与植物幼年期向成年期的转变、营养生长向生殖生长的转变、花发育、孢子发生、叶片和根发育、逆境响应等多个过程，在植物的生长发育过程中起着非常重要的作用。探究白桦中BpSPL6基因启动子区的顺式作用元件，以及该启动子在正常和胁迫条件下的表达模式，可为进一步研究BpSPL6基因的功能提供参考，也可为了解白桦的抗逆机制提供依据。 方法 以本实验室组培白桦的总DNA为模板，经PCR克隆了BpSPL6基因上游1 703 bp的启动子序列，用PLACE和Plant CARE在线软件分析启动子区的顺式作用元件。构建了BpSPL6基因启动子驱动GUS报告基因的植物表达载体并转化拟南芥，探究其组织表达特性和胁迫条件下的表达模式。 结果 PCR成功克隆了BpSPL6基因上游1 703 bp的启动子序列，对启动子区的顺式作用元件预测发现除了含有核心启动元件TATA-box、CAAT-box外，还包括2种特异组织表达元件（根、花粉），10种激素响应元件（生长素、赤霉素、水杨酸、脱落酸），4种脱水响应元件等。对转基因拟南芥进行GUS染色结果表明，BpSPL6基因启动子驱动的GUS基因在转基因拟南芥中的表达具有时空特异性。在拟南芥的整个发育过程中，BpSPL6基因启动子驱动GUS基因在真叶叶片中表达，但是表达部位不同。随着叶片的生长，首先在叶片的顶端表达，随后扩展到叶片的叶脉并直至整个叶片，并且表达量逐渐升高。同时BpSPL6基因启动子驱动的 GUS 基因在拟南芥营养生长时期的根部都有表达。并且经氯化钠和甘露醇胁迫后其表达量降低。对比两种胁迫，受到氯化钠胁迫后GUS基因的表达量变化更大，说明对氯化钠胁迫的响应更加强烈。 结论 BpSPL6基因可能参与了植物的叶片、根发育以及对盐和干旱胁迫的响应。 Abstract:Objective SPL is a plant-specific transcription factor and plays crucial roles in the growth and development of plants. It participates in multiple processes such as plant phase transformation, flower development, sporogenesis, leaf and root development, and stress response. Exploring the cis-acting elements of the promoter region of the BpSPL6 gene, as well as the expression pattern under normal and stress conditions, this paper provides a reference for further study of the function of BpSPL6 gene and also provides a basis for understanding the stress resistance mechanism of Betula platyphylla. Method Using the total DNA of tissue-cultures B. platyphylla in this laboratory as a template, the 1 703 bp promoter sequence of BpSPL6 gene was cloned by PCR. The cis-elements of the promoter region were analyzed using PLACE and Plant CARE web tools. We constructed the BpSPL6 gene promoter-driven GUS expression vector, which was transformed into Arabidopsis thaliana. Transgenic A. thaliana was subjected to stress experiments, and GUS staining was used to analyze the expression pattern of the BpSPL6 gene promoter. Result A 1 703 bp promoter sequence of BpSPL6 gene was cloned from B. platyphylla genomic DNA. Sequence analysis showed that the BpSPL6 gene promoter included core promoter elements TATA-box and CAAT-box, as well as cis-elements for specific parts (root and pollen), hormone response elements (auxin, gibberellin, salicylic acid, abscisic acid) and dehydration response elements. The GUS staining results of transgenic A. thaliana showed that the expression of GUS gene driven by BpSPL6 gene promoter had spatiotemporal specificity. Throughout the development of A. thaliana, the GUS gene driven by BpSPL6 gene promoter was expressed in euphylla, but the expression sites were different. As the leaf grows, it is first expressed at the tip of the leaf, then expands to the leaf vein and reaches the entire leaf, and the expression level gradually increases. At the same time, the GUS gene driven by BpSPL6 gene promoter is also expressed in the roots during vegetative growth. When transgenic A. thaliana was subjected to sodium chloride and mannitol stress, the expression level of GUS gene driven by BpSPL6 gene promoter decreased compared with control. Comparing the two stresses, the expression level of GUS gene changed more after being subjected to sodium chloride stress, indicating that the response to sodium chloride stress was stronger. Conclusion BpSPL6 gene may be involved in plant leaf and root development and response to salt and drought stress. -
Key words:
- Betula platyphylla /
- SPL /
- promoter /
- cis-element /
- GUS staining /
- salt stress /
- drought stress
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图 1 BpSPL6基因启动子的克隆
M. DL-5000 Marker; 1. BpSPL6基因启动子扩增产物。M, DL-5000 Marker; 1, amplified production of BpSPL6 promoter.
Figure 1. Cloning of BpSPL6 promoter by PCR
图 2 BpSPL6基因启动子核苷酸序列多重比对
1-BpSPL6-promoter. 测序结果；2-BpSPL6-promoter. 白桦基因组数据库中启动子序列；3-BpSPL6-promoter. 欧洲白桦基因组数据库中启动子序列。1-BpSPL6-promoter, sequencing results; 2-BpSPL6-promoter, BpSPL6 promoter sequences in Betula platyphylla genome database; 3-BpSPL6-promoter, BpSPL6 promoter sequences in Betula pendula genome database.
Figure 2. Multi-alignment of nucleotide sequences of BpSPL6 promoter
图 3 白桦BpSPL6基因启动子序列及顺式作用元件分布
方框及彩色表示预测的顺势作用元件序列；方框下文字表示对应元件名称；图片下部注释了彩色序列对应的元件名称；下划线表示启动子上游未克隆序列；双下划线并加粗表示起始密码子位置。The box and color represent the predicted cis-elements; text under the box represents the corresponding cis-element name; the cis-element names corresponding to the color sequence are annotated at the bottom of the picture; the underline indicates the uncloned sequence upstream of the promoter; the double underline and bold indicate the position of start codon.
Figure 3. Promoter sequence and cis-element distribution of BpSPL6 gene promoter for B. platyphylla
图 4 大肠杆菌重组质粒的菌液PCR检测
M. DL-5000 Marker；1. 阴性对照（水对照）；2 ~ 3. 大肠杆菌转化子。M, DL-5000 Marker; 1, negative control（water control）; 2−3, Escherichia coli transformant.
Figure 4. PCR result of positive Escherichia coli clone
图 5 转基因拟南芥的检测
M. DL-5000 Marker；1. 阳性对照；2 ~ 6. 转基因单株；7. 阴性对照（野生型拟南芥）。M, DL-5000 Marker; 1, positive control; 2−6, transgenic lines; 7, negative control（wild type A. thaliana）.
Figure 5. PCR detection of transgenic A. thaliana
图 6 营养生长期的转基因拟南芥GUS染色
A ~ C. 生长7 d的拟南芥；A. 整株幼苗；B. 真叶；C. 根。D ~ G. 生长10 d的拟南芥；D. 整株幼苗；E. 真叶；F. 下胚轴；G. 根。A−C, A. thaliana seedling grown for 7 days; A, whole seedling; B, euphylla; C, root. D−G, A. thaliana seedling grown for 10 days; D, whole seedling; E：euphylla；F, hypocotyl; G, root.
Figure 6. Staining of GUS in transgenic A. thaliana in vegetative phase
图 7 生殖生长期的转基因拟南芥GUS染色
A ~ G. 生长25 d的拟南芥；A. 整株幼苗；B. 成熟叶片；C. 幼嫩叶片；D. 幼嫩叶片和花；E. 花；F. 幼果；G. 根。A−G, A. thaliana seedling grown for 25 days; A, whole seedling; B, mature leaf; C, young leaf; D, young leaf and flower; E, flower; F, young fruit; G, root.
Figure 7. Staining of GUS in transgenic A. thaliana in reproductive phase
图 8 不同非生物胁迫处理下转基因拟南芥的GUS染色
A. 整株幼苗；B. 成熟叶片；C. 幼嫩叶片；D. 根；E. 下胚轴。A, whole seedling; B, mature leaf; C, young leaf; D, root; E, hypocotyl.
Figure 8. Staining of GUS in transgenic A. thaliana under different abiotic stress treatments
表 1 启动子顺式作用元件分析
Table 1. Analysis of promoter cis-elements
元件名称 Element name 基序序列 Motif sequence 个数 Number 生物学功能 Biological function TATA-box TATA 6 核心启动元件 Core actuating element CAAT-box CAAT 22 启动子增强区保守元件 Conservative element of promoter enhance region GT1-motif GGTTAAT 5 光响应元件 Element involved in light responsiveness Box 4 ATTAAT 3 光响应元件 Element involved in light responsiveness GA-motif ATAGATAA 2 光响应元件 Element involved in light responsiveness AT1-motif AATTATTTTTTATT 1 光响应元件 Element involved in light responsiveness AuxRR-core GGTCCAT 2 生长素响应元件 Element involved in auxin responsiveness TGA-element AACGAC 1 生长素响应元件 Element involved in auxin responsiveness CATATGGMSAUR CATATG 4 生长素响应元件 Auxin response element NTBBF1ARROLB ACTTTA 4 生长素响应元件 Auxin response element GARE2OSREP1 TAACGTA 1 赤霉素响应元件 Gibberellin response element WRKY71OS TGAC 12 赤霉素响应元件 Gibberellin response element P-box CCTTTTG 1 赤霉素响应元件 Gibberellin response element WBOXATNPR1 TTGAC 4 水杨酸响应元件 Element involved in salicylic acid responsiveness DPBFCOREDCDC3 ACACNNG 3 脱落酸响应元件 Element involved in abscisic acid responsiveness ABRE ACGTG 1 脱落酸响应元件 Element involved in abscisic acid responsiveness MYB1AT WAACCA 5 脱水响应元件 Dehydration response element MYCCONSENSUSAT CANNTG 12 脱水及寒冷响应元件 Dehydration and cold response element ACGTATERD1 ACGT 4 脱水响应元件 Dehydration response element MYBCORE CNGTTR 1 脱水响应元件 Dehydration response element WBOXNTERF3 TGACY 7 伤害响应元件 Damage response element DOFCOREZM AAAG 33 伤害响应元件 Damage response element CCAATBOX1 CCAAT 4 热激信号响应元件 Heat shock signal response element POLLEN1LELAT52 AGAAA 14 花粉特异表达的顺式作用元件 Cis-acting element for pollen specific expression ROOTMOTIFTAPOX1 ATATT 10 与根相关的顺式作用元件 Cis-element element in root 注：基序序列：N = A/T/G/C，R = A/G，W = A/T，Y = C/T。Notes: motif sequences: N = A/T/G/C, R = A/G, W = A/T, Y = C/T. 
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