Cloning and Expression Analysis of Furostanol Glycoside 26-O-β-glucosidase Gene in Anoectochilus roxburhii
-
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.
下载: 导出CSV
-
[1] 刘星, 余江丽, 刘敏, 等. 近10年甾体皂苷的生物活性研究进展 [J]. 中国中药杂志, 2015, 40(13):2518−2523.LIU X, YU J L, LIU M, et al. Research progress of bioactivity of steroidal saponins in recent ten years [J]. China Journal of Chinese Materia Medica, 2015, 40(13): 2518−2523.(in Chinese) [2] 黄圆圆, 刘大会, 彭华胜, 等. 15种重楼属植物中8种甾体皂苷的含量测定 [J]. 中国中药杂志, 2017, 42(18):3443−3451.HUANG Y Y, LIU D H, PENG H S, et al. Determination of eight steroidal saponins in 15 kinds of genus Paris [J]. China Journal of Chinese Materia Medica, 2017, 42(18): 3443−3451.(in Chinese) [3] 旷湘楠, 刘时乔. 麦冬中甾体皂苷类化学成分研究 [J]. 广州化工, 2017, 45(22):85−87. doi: 10.3969/j.issn.1001-9677.2017.22.030KUANG X N, LIU S Q. Steroidal saponins from Tuber of Ophiopogon japonicas [J]. Guangzhou Chemical Industry, 2017, 45(22): 85−87.(in Chinese) doi: 10.3969/j.issn.1001-9677.2017.22.030 [4] 李琳玉, 刘星, 周梦, 等. 小果菝葜根茎的甾体皂苷类化学成分研究 [J]. 中药材, 2017, 40(9):2084−2088.LI L Y, LIU X, ZHOU M, et al. Steroidal saponins from the rhizomes of Smilax davidiana [J]. Journal of Chinese Medicinal Materials, 2017, 40(9): 2084−2088.(in Chinese) [5] 金明, 仙靓, 孙丽娜, 等. 盾叶薯蓣中甾体皂苷的分离与结构鉴定 [J]. 西北药学杂志, 2017, 32(4):395−399. doi: 10.3969/j.issn.1004-2407.2017.04.001JIN M, XIAN L, SUN L N, et al. Isolation and structure identification of steroid saponins from Dioseorea zingiberensis C.H.Wright [J]. Northwest Pharmaceutical Journal, 2017, 32(4): 395−399.(in Chinese) doi: 10.3969/j.issn.1004-2407.2017.04.001 [6] 尹艳, 关红雨, 张夏楠. 甾体皂苷生物合成相关酶及基因研究进展 [J]. 天然产物研究与开发, 2016, 28(8):1332−1336.YIN Y, GUAN H Y, ZHANG X N. Review on enzymes and genes related to the biosynthesis of steroidal saponins [J]. Natural Product Research and Development, 2016, 28(8): 1332−1336.(in Chinese) [7] 黄楚君, 蔡金艳, 倪俊, 等. 束花石斛化学成分研究 [J]. 广东药学院学报, 2016, 32(3):279−281.HUANG C J, CAI J Y, NI J, et al. Study on chemical constituents from Dendrobium chrysanthum Wall. ex Lindl [J]. Journal of Guangdong Pharmaceutical University, 2016, 32(3): 279−281.(in Chinese) [8] 刘海, 杨建琼, 马华谋, 等. 吉祥草中甾体皂苷成分及其抗肿瘤活性研究 [J]. 中药新药与临床药理, 2015, 26(3):348−351.LIU H, YANG J Q, MA H M, et al. Analysis of steroidal saponins from reineckia carnea and their antitumor activities [J]. Traditional Chinese Drug Research and Clinical Pharmacology, 2015, 26(3): 348−351.(in Chinese) [9] BARILE E, BONANOMI G, ANTIGNANI V, et al. Saponins from Allium minutiflorum with antifungal activity [J]. Phytochemistry, 2007, 68(5): 596−603. doi: 10.1016/j.phytochem.2006.10.009 [10] INOUE K, SHIBUYA M, YAMAMOTO K, et al. Molecular cloning and bacterial expression of a cDNA encoding furostanol glycoside 26-O-β-glucosidase of Costus speciosus [J]. FEBS Letters, 1996, 389(3): 273−277. doi: 10.1016/0014-5793(96)00601-1 [11] 王亮, 游松, 蒋雅红, 等. 利用重组F26G酶实现呋甾皂苷向螺甾皂苷的体外生物转化 [J]. 中国药物化学杂志, 2001, 11(6):326−328. doi: 10.14142/j.cnki.cn21-1313/r.2001.06.004WANG L, YOU S, JIANG Y H, et al. Bioconversion in vitro from furostanol glycoside to spirostanol glycoside catalyzed by recombinated F26G [J]. Chinese Journal of Medicinal Chemistry, 2001, 11(6): 326−328.(in Chinese) doi: 10.14142/j.cnki.cn21-1313/r.2001.06.004 [12] NAKAYASU M, KAWASAKI T, LEE H J, et al. Identification of furostanol glycoside 26-O-β-glucosidase involved in steroidal saponin biosynthesis from Dioscorea esculenta [J]. Plant Biotechnology, 2015, 32(4): 299−308. doi: 10.5511/plantbiotechnology.15.1023b [13] 张超, 吴建国, 易骏, 等. HPLC-ELSD法测定三种植物基原金线莲的金线莲苷含量 [J]. 食品工业科技, 2017, 38(2):75−78.ZHANG C, WU J G, YI J, et al. Content determination of kinsenoside in Jin-Xian-Lian from three Anoectochilus species by HPLC-ELSD [J]. Science and Technology of Food Industry, 2017, 38(2): 75−78.(in Chinese) [14] 施满容, 龚林光, 陆志平, 等. 不同地区野生金线莲有效成分含量的比较 [J]. 安徽农学通报, 2016, 22(24):107−110. doi: 10.3969/j.issn.1007-7731.2016.24.047SHI M R, GONG L G, LU Z P, et al. The comparison of effective composition content in different localities of wild Anoectochilus roxburhii [J]. Anhui Agricultural Science Bulletin, 2016, 22(24): 107−110.(in Chinese) doi: 10.3969/j.issn.1007-7731.2016.24.047 [15] 张锦文, 唐菲, 张小琼, 等. 高效液相色谱法测定金线莲中金线莲苷的含量 [J]. 中国医院药学杂志, 2011, 31(4):261−263.ZHANG J W, TANG F, ZHANG X Q, et al. Determination of the content of kinsenoside in Anoectochilus roxburhii (Wall.) Lindl by HPLC [J]. Chinese Journal of Hospital Pharmacy, 2011, 31(4): 261−263.(in Chinese) [16] 王勇, 陈硕, 卢端萍, 等. 金线莲化学成分的研究 [J]. 中草药, 2017, 48(13):2619−2624.WANG Y, CHEN S, LU D P, et al. Chemical constituents of Anoectochilus roxburhii [J]. Chinese Traditional and Herbal Drugs, 2017, 48(13): 2619−2624.(in Chinese) [17] 吴丽丽, 梁燕, 许光辉. 金线莲化学成分、药理作用及临床应用研究概述 [J]. 海峡药学, 2014, 26(10):34−37. doi: 10.3969/j.issn.1006-3765.2014.10.013WU L L, LIANG Y, XU G H. Advances on investigation of chemical components, pharmacological activities and clinical applications of Anoectochilus roxburhii [J]. Strait Pharmaceutical Journal, 2014, 26(10): 34−37.(in Chinese) doi: 10.3969/j.issn.1006-3765.2014.10.013 [18] 肖小华, 林彩霞, 吴序栎, 等. 金线莲的化学成分及生物活性研究进展 [J]. 现代食品科技, 2018, 34(5):267−275.XIAO X H, LIN C X, WU X L, et al. Research advance on chemical constituents and biological activities of Anoectochilus roxburhii [J]. Modern Food Science and Technology, 2018, 34(5): 267−275.(in Chinese) [19] TAMURA K, STECHER G, PETERSON D, et al. MEGA6: molecular evolutionary genetics analysis version 6.0 [J]. Molecular Biology and Evolution, 2013, 30(12): 2725−2729. doi: 10.1093/molbev/mst197 [20] 林江波, 王伟英, 李海明, 等. 中国水仙锌指蛋白NtPLATZ1的克隆与表达分析 [J]. 西北农林科技大学学报(自然科学版), 2016, 44(10):165−170.LIN J B, WANG W Y, LI H M, et al. Cloning and expression analysis of NtPLATZ1 gene from Narcissus tazetta var. chinensis [J]. Journal of Northwest A & F University(Natural Science Edition), 2016, 44(10): 165−170.(in Chinese) [21] 林江波, 王伟英, 邹晖, 等. 金线莲3个持家基因表达稳定性分析 [J]. 福建农业学报, 2018, 33(11):1125−1129.LIN J B, WANG W Y, ZOU H, et al. Expression stabilities of three housekeeping genes of Anoectochilus roxburhii [J]. Fujian Journal of Agricultural Sciences, 2018, 33(11): 1125−1129.(in Chinese) [22] MORANT A V, JØRGENSEN K, JØRGENSEN C, et al. β-Glucosidases as detonators of plant chemical defense [J]. Phytochemistry, 2008, 69(9): 1795−1813. doi: 10.1016/j.phytochem.2008.03.006 [23] CHEN Y N, WANG L, REN J, et al. The selective biotransformation of furostanol glycosides and their analogs by recombined F-26-O-β-glucosidase [J]. Asian Journal of Traditional Medicines, 2009, 4(1): 7−13. -
点击查看大图
计量
- 文章访问数: 1254
- HTML全文浏览量: 637
- PDF下载量: 15
- 被引次数: 0
