Cloning and Analyzing of AP3-3 and Its Promoter from Dendrobium officinale
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摘要:
目的 AP3-3属于MADS-box基因家族B类基因，参与兰科植物花被和唇瓣的形成，克隆该基因并进行启动子分析可以进一步研究该基因的生物学功能及其启动子调控作用机制。 方法 使用RT-PCR和常规PCR技术克隆铁皮石斛DoAP3-3基因及其启动子序列，进行生物信息学分析，构建启动子缺失片段与GUS基因融合表达载体，农杆菌介导转化铁皮石斛原球茎，进行瞬时表达。 结果 DoAP3-3基因cDNA长度为675 bp，编码蛋白质的分子式为C1129H1803N333O347S12，分子量25.98 kDa，pI为 8.71，不稳定性指数40.14，GRAVY为−0.823，不存在跨膜区域，亚细胞定位预测得分为细胞核87.0%、线粒体8.7%、细胞质4.3%。启动子片段长度1885 bp，顺式作用元件含有大量的光响应元件等；3个启动子片段均可驱动GUS基因表达，表达强度为−1885～0 bp＞−1604～0 bp＞−750～0 bp。 结论 DoAP3-3蛋白具有碱性、亲水性和不稳定性，无跨膜结构域，亚细胞定位于细胞核中。DoAP3-3启动子可能受光照、植物激素、MYB转录蛋白等多个因素调控，具备启动活性，且随启动子缺失长度减少呈现增加趋势。 Abstract:Objective AP3-3 of class B gene of MADS box family that involves in the formation of perianth and labellum of Dendrobium officinale was cloned to study the biological function, and the promoter analyzed to decipher the regulation mechanism. Method Sequences of AP3-3 and promoter of D. officinale were cloned by RT-PCR and conventional PCR, and bioinformatics analyzed. A fusion expression vector of promoter-deleted fragments and GUS gene was constructed. Agrobacterium tumefaciens-mediated transformation was used to transform protocorm of D. officinale for the transient expression. Result The cDNA length of DoAP3-3 was 675 bp with an encoded formula of C1129H1803N333O347S12, a molecular weight of 25.98 kDa, a PI of 8.71, an instability index of 40.14, and GRAVY of −0.823. There was no transmembrane region detected in the protein. The predicted score of subcellular localization was 87.0% in nucleus, 8.7% in mitochondria, and 4.3% in cytoplasm. The 1885 bp promoter fragment had a cis acting element containing a significant number of photo-responsive elements among others. The 3 promoter fragments could drive GUS with an order of expression intensity of −885–0 bp＞−1 604–0 bp＞−750–0 bp. Conclusion The predicted DoAP3-3 was an alkali, hydrophilic, and unstable protein with no transmembrane domain and a subcellular localization in the nucleus. The DoAP3-3 promoter might be regulated by light, plant hormones, MYB transcription protein, etc., and exhibited activities that increased with decreasing deletion length. -
Key words:
- Dendrobium officinale /
- AP3-3 /
- cDNA /
- promoter /
- cloning /
- sequence analysis
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图 1 PCR扩增电泳
A：DoAP3-3扩增电泳图（M：DNA marker DL5000）；B： DoAP3-3启动子扩增电泳图（M：DNA marker DL6000）。
Figure 1. Electrophoresis of PCR amplification
A: amplification electrophoresis of DoAP3-3 (M: DNA marker DL5000); B: amplification electrophoresis of DoAP3-3 promoter (M: DNA marker DL6000).
图 2 DoAP3-3氨基酸序列与其他植物同源序列的系统进化树
Figure 2. Phylogenetic tree of DoAP3-3 and homologous amino acid sequences in other plants
图 3 DoAP3-3与其他植物AP3-3氨基酸序列的同源比对
Figure 3. Homologous alignment of amino acid sequences between DoAP3-3 and AP3-3 in other plants
图 6 启动子融合GUS重组载体构建
A：启动子扩增电泳图（M：DNA marker DL6000；1：DoAP3-3-1885；2：DoAP3-3-1604；3：DoAP3-3-750）；B：菌落PCR电泳（M：DNA marker DL6000；1：pB-DoAP3-3-A；2：pB-DoAP3-3-B；3：pB-DoAP3-3-C）。
Figure 6. Construction of fusion recombinant vector of promoter and GUS
A: promoter amplification electrophoresis (M: DNA marker DL6000; 1: DoAP3-3-1885; 2: DoAP3-3-1604; 3: DoAP3-3-750); B: colony PCR electrophoresis (M: DNA marker DL6000; 1: pB-DoAP3-3-A; 2: pB-DoAP3-3-B; 3: pB-DoAP3-3-C).
图 7 转基因铁皮石斛原球茎GUS染色
A：pB-DoAP3-3-A；B：pB-DoAP3-3-B；C：pB-DoAP3-3-C；D：阴性对照（野生型）；E： 阳性对照［pBWA(V)HG］。
Figure 7. GUS stained protocorm of transgenic D. officinale
A: pB-DoAP3-3-A; B: pB-DoAP3-3-B; C: pB-DoAP3-3-C; D: negative control (wild type); E: positive control [pBWA(V)HG].
表 1 PCR引物信息
Table 1. PCR Primer information
引物名称
Primer name引物序列（5′→3′）
Primer sequence用途
Usage产物大小
Amplicon size/bpDoAP3-3 (F) TATCTTCCCCCTCCCCAT 基因克隆Gene cloning 675 DoAP3-3 (R) ATCTTCGTCTCGCTTGA DoAP3-3-promoter (F) CGCCGTTACCTGCGTCGTTC 启动子克隆Promoter cloning 1885 DoAP3-3-promoter (R) CCTGATCACTTCTTCTCCTC DoAP3-3-promoter-A (F) CAGTGGTCTCATAGACGCCGTTACCTGCGTCGTTC 启动子表达载体构建Construction of expression vector driven by DoAP 3-3 promoter 1885 DoAP3-3-promoter-B (F) CAGTGGTCTCATAGAGTTGGACAAAACTTTGAGAT 1604 DoAP3-3-promoter-C (F) CAGTGGTCTCATAGACAGTGATTTAAGGGGAATGG 750 DoAP3-3-promoter (R) CAGTGGTCTCAGTTGCCTGATCACTTCTTCTCCTC GUS-promoter-A (F) GAAGTTGAAGACCAATAAACT 检测启动子是否与GUS连接Detection of whether the DoAP3-3 promoter is connected to GUS 630 GUS-promoter-B (F) GCTGATGAATTTTTAAAATTCT 568 GUS-promoter-C (F) GATATTATGAAGCGTGAATG 374 GUS-promoter (R) ATAAAAAGAGAAAAGGGTCCTAACC 下划线为Eco31 I酶识别位点。
Recognition site of Eco31 I enzyme is underlined.
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表 2 DoAP3-3启动子顺式作用元件预测分析
Table 2. Predicted DoAP3-3 promoter cis-acting element
顺式作用元件
Cis-acting
elements位置(+/−链)
Position from
ATG/bp核心序列
Core sequence功能
FunctionAAGAA-motif 33 GAAAGAA 涉及脱落酸反应的顺式作用元件 Cis-acting element involved in abscisic acid reaction ACE 1479 CTAACGTATT 参与光响应的顺式作用元件 Cis-acting element involved in light responsiveness Box 4 1519, 898, 1331,
450, 1406, 708ATTAAT 参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness CAT-box 674 GCCACT 与分生组织表达相关的顺式作用调控元件 Cis-acting regulatory element related to meristem expression GATA-motif 270 AAGGATAAGG 光响应元件的一部分 Part of a light responsive element GT1-motif 1115, 1652 GGTTAA 光响应元件 Light responsive element GTGGC-motif 1636 CATCGTGTGGC 光响应元件的一部分 Part of a light responsive element I-box 268 GATAAGGCG 光响应元件的一部分 Part of a light responsive element MBS 19 CAACTG MYB结合位点参与干旱诱导 MYB binding site involved in drought-inducibility MRE 1655 AACCTAA MYB结合位点参与光响应 MYB binding site involved in light responsiveness P-box 1708 CCTTTTG 赤霉素反应元件 Gibberellin-responsive element TATA-box 1360, 1003, 1728,
1001, 977, 917, 336TATATA 转录起始核心元件 Core promoter element of transcription start TGA-element 1866 AACGAC 生长素反应元件 Auxin-responsive element W box 1797 TTGACC 水杨酸响应元件 Salicylic acid-responsive element
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