Cloning and Expression Analysis of Furostanol Glycoside 26-O-β-glucosidase Gene in Anoectochilus roxburhii
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摘要:
目的 克隆金线莲（Anoectochilus roxburhii）ArF26G基因的cDNA全长，分析该基因在金线莲茎、叶中的表达模式，以期为进一步了解金线莲甾体皂苷的生物合成代谢调控机制提供参考。 方法 利用RACE技术克隆金线莲ArF26G基因cDNA全长，以pET-28a（+）为载体构建原核表达载体并诱导表达，采用荧光定量PCR方法分析不同温度（5、15、25、35℃）和种植时间（1–6个月）ArF26G基因在茎和叶中的表达水平。 结果 ArF26G基因的cDNA全长1 982 bp，含有1个1 764 bp的ORF，编码587个氨基酸，编码蛋白具有Glycosyl hydrolase 1（GH1）superfamily蛋白保守区，定位于叶绿体，N端33个氨基酸残基为叶绿体转运肽，分子量为66.48 kD，理论等电点（pI）为5.31，不稳定系数为37.92，属稳定蛋白。构建了pET-28a-ArF26G原核表达载体，并在大肠杆菌（E. coli）BL21（DE3）中成功诱导表达。荧光定量PCR分析结果显示，ArF26G基因在茎中的表达量极显著高于叶，25℃处理表达量最高。金线莲种植时间达4个月时，ArF26G基因在茎中的表达量是25℃处理组培苗茎的11.9倍，种植5个月达到23.3倍。 结论 本研究克隆了金线莲ArF26G基因cDNA全长，其表达量在25℃以及种植5个月时表达量最高。 -
关键词:
- 金线莲 /
- 呋甾皂苷26-O-β-糖苷酶 /
- 甾体皂苷 /
- 基因表达
Abstract:Objective In order to further understand the regulatory mechanism of steroidal saponins biosynthesis and metabolism of Anoectochilus roxburhii, the full-length cDNA of ArF26G gene was cloned and its expression pattern in the stem and leaf was analyzed. Method The full-length cDNA of ArF26G gene was cloned by RACE. Prokaryotic expression vector was constructed with pET-28a (+), and induced by IPTG. The expression levels of ArF26G gene in stem and leaf of Anoectochilus roxburhii at different temperatures and planting times were analyzed by fluorescence quantitative PCR. Result The full-length cDNA of ArF26G gene is 1 982 bp with an 1 764 bp ORF, which encoded a protein of 587 amino acids containing a conservative domain of Glycosyl hydrolase 1 (GH1) superfamily. ArF26G is located in chloroplast and a putative chloroplast transit peptide of 33 amino acid residues at the N-terminus. The molecular weight of ArF26G protein is 66.48 kD, and the theoretical isoelectric (pI) is 5.31, and the instability coefficient is 37.92, it is a stable protein. Prokaryotic expression vector pET-28a-ArF26G was constructed and induced successfully by IPTG in E. coli BL21 (DE3). The results of fluorescence quantitative PCR analysis showed that the expression of ArF26G in stem was significantly higher than that in leaf, and the highest expression of tissue culture seedlings was at 25℃. When the planting time was up to 4 months and 5 months, the expression of ArF26G gene in the stem was 11.9 times and 23.3 times as much as that in the stem of tissue culture seedlings treated at 25℃ respectively. Conclusion In this study, the full-length cDNA of ArF26G gene was cloned. The expression of ArF26G gene was the highest at 25℃ or planting for 5 months. -
图 1 PCR电泳结果
注：M：分子量标记；A：3′RACE；B：开放阅读框。
Figure 1. PCR products by electrophoresis
Note: M: DNA marker (100-2000); A: 3′RACE; B: ORF.
图 2 金线莲ArF26G基因cDNA序列及其推导的氨基酸序列
Figure 2. The cDNA and its deduced amino acid sequence of ArF26G from Anoectochilus roxburhii
图 3 ArF26G蛋白的二级结构预测
注：大写字母为氨基酸序列；小写字母为二级结构；c表示无规则卷曲；h表示α-螺旋；e表示β-折叠。
Figure 3. Predicted secondary structure of ArF26G
Note: capital letters show amino acid sequence; small letters show structure of amino acid chain; c shows random coil; h shows α-helix; e shows β-extended.
图 4 ArF26G与其他物种GH1结构域蛋白的系统进化树
Figure 4. Phylogenetic tree of ArF26G and GH1 domain proteins of other plants
图 5 ArF26G基因在大肠肝菌中表达的SDS-PAGE电泳
注：M：分子量标记；1：BL21诱导；2：携带pET-28a-ArF26G BL21未诱导；3：携带pET-28a-ArF26G BL21诱导。
Figure 5. SDS-PAGE of ArF26G expressed in Esherichia coli.
Note: M: Marker; 1: induction (BL21); 2: non-induction (BL21 contained pET-28a-ArF26G); 3: induction (BL21 contained pET-28a-ArF26G).
图 6 不同处理温度和种植时间ArF26G在茎和叶中的相对表达量
注：不同大写字母表示在0.01水平上差异显著。
Figure 6. Relative expression of ArF26G in stem and leaf under different treatment temperature and planting time.
Note: Different capital letters indicate significant difference at 0.01 level.
表 1 PCR引物及其序列
Table 1. PCR primers and sequences
引物名称
Primer name引物序列（5′-3′）
Primer sequence（5′-3′）3F26-F1 GGTTTGTGCCATACTCCGATAG 3F26-F2 GAGTGGTGACTTGAACAATCTG dT-adapt CTGATCTAGAGGTACCGGATCCTTTTTTTTTTTTTTTTT adapt CTGATCTAGAGGTACCGGATCC F26-F TTTGGATCCATGTCTCTGTCCCTGTCTTTTA F26-R GAAGAGCTCTCAATACAATGCAGTGACGCT F26G-F CCAATCCAGCACAAGTAACA F26G-R CCATTGGTAAATACTGACGG ArACT-F GCATAGCCTTCATAGATGGG ArACT-R GAGGACATTCAGCCACTTG 注：下划线碱基为酶切位点。
Note：The underlined bases were the enzyme site.
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