芍药<i>ACS</i>基因的克隆及其蛋白质原核表达

doi:10.7668/hbnxb.20193096

摘要/Abstract

摘要：

为了研究芍药ACS基因的功能,应用RACE技术从芍药切花品种桃花飞雪中克隆了PlACS基因cDNA序列,利用生物信息学方法对其编码的蛋白质进行了综合分析;以pET32a为骨架,构建了PlACS基因的原核表达载体,建立了PlACS蛋白高效的原核表达体系。结果表明,PlACS cDNA全长1 752 bp(GenBank登录号JX512359),共编码492个氨基酸,具有7个保守结构域及活性位点K₂₇₈;分子进化分析表明,PlACS和牡丹ACS亲缘关系最近;二级结构预测发现,PlACS蛋白由α-螺旋、β-折叠、β-转角和无规则卷曲组成,四者比例分别为40.04%,16.26%,6.91%和36.79%;同源建模显示,PlACS蛋白以同源二聚体形式存在。PlACS蛋白最佳诱导表达条件为基因工程菌菌体密度A₆₀₀达0.2时,在菌液中加入终浓度为0.1 mmol/L的IPTG,在37 ℃诱导2 h。PlACS重组蛋白高效原核表达体系为后续通过变复性获得有生物学活性的PlACS蛋白及其酶学功能鉴定奠定基础。

关键词: 芍药, PlACS, 基因克隆, 序列分析, 原核表达

Abstract:

In order to explore the function of ACS gene in herbaceous peony,a full-length cDNA sequence of PlACS cDNA in Paeonia lactiflora was obtained,RACE technique and bioinformatic methods were used to analyze the protein sequence which it encoded.The CDS of PlACS was subcloned,the prokaryotic expression vector of PlACS was constructed based on pET32a vector,and then the highly efficient prokaryotic expression system was established.The results showed that the total length of PlACS cDNA(GenBank accession JX512359)was 1 752 bp,which encoded 492 amino acids.Seven conserved regions and active sites K₂₇₈ were detected in PlACS protein.Phylogenetic tree analysis showed that PlACS was highest homological with ACS of P.suffruticosa.PlACS protein was determined structurally to be 40.04% α-helix,16.26% β-extended strand,6.91% β-turn and 36.79% random coil.Protein 3D structure homology modeling predicted that PlACS existed as homodimers.The optimal expression condition of PlACS protein was that when the cell density of genetic engineering strain A₆₀₀ reached 0.2,IPTG with a final concentration of 0.1 mmol/L was added,and the recombinant protein was expressed for two hours at 37 ℃.It was of great significance to acquire PlACS recombinant protein with biological activity by denaturation & renaturation and identify its enzymatic activity in vitro.

Key words: Paeonia lactiflora, PlACS, Gene cloning, Sequence analysis, Prokaryotic expression

肖士奎, 李芳, 张文婷, 吕淑芳, 史国安, 吴疆, 范丙友. 芍药ACS基因的克隆及其蛋白质原核表达[J]. 华北农学报, 2022, 37(5): 36-44. doi: 10.7668/hbnxb.20193096.

XIAO Shikui, LI Fang, ZHANG Wenting, LÜ Shufang, SHI Guoan, WU Jiang, FAN Bingyou. Cloning and Prokaryotic Expression of ACS Gene from Paeonia lactiflora Pall.[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 36-44. doi: 10.7668/hbnxb.20193096.

http://www.hbnxb.net/CN/Y2022/V37/I5/36

图/表 14

表1 引物序列

Tab.1 Primer sequences

引物名称 Primer name	引物序列(5'—3') Primer sequences(5'—3')	作用 Function
PlACS-F	CGGGATCCDTWYCARGAYTAYCAYGG	PlACS保守区扩增
PlACS-R	AACTGCAGGAIARRCTRKAIAVDAYRTG	PlACS保守区扩增
PlACS-5'-1^st	TTCCGCAATGCTAATGAAACGAGGCA	PlACS基因5'-RACE第1轮
PlACS-5'-2^nd	GACAGTGGCCGCGTAAATCTCATC	PlACS基因5'-RACE第2轮
PlACS-3'	TCAACTGCTTCCCGTTCAATGCGAC	PlACS基因3'-RACE
PlACS-OF	CGCGGATCCATGGATTCATGTCCACAGATC	原核表达载体构建
PlACS-OR	CCCAAGCTTTTAAGCTCGAACAAGGGGTGAT	原核表达载体构建

图1 PlACS基因的PCR扩增 A.保守序列;B.3'-RACE;C.5'-RACE第1轮;D.5'-RACE第2轮。

Fig.1 PCR amplification of PlACS A.Conservative sequence;B.3'-RACE;C.5'-RACE round 1;D.5'-RACE round 2.

图2 PlACS基因核苷酸及编码的氨基酸序列 ATG.起始密码子;TAA.终止密码子;小写字母.5'-UTR和3'-UTR;下划线.可能的磷酸化位点。

Fig.2 Nucleotides and encoded amino acids sequences of PlACS ATG.The start codon;TAA.The stop codon;The lowercase letters.5'-UTR and 3'-UTR;The underline.Possible phosphorylation sites.

图3 ACS蛋白多序列比对红色框.ACS蛋白的保守结构域;红色箭头.活性位点K₂₇₈。

Fig.3 Multiple sequence alignment of ACS proteins Red box.Conservative domains of ACS proteins;Red arrow.Active site K₂₇₈.

图4 PlACS蛋白与其他物种ACS蛋白的系统进化树

Fig.4 Phylogenetic tree of PlACS and previously reported ACS proteins

图5 PlACS蛋白二级结构分析 A.α-螺旋;B.β-折叠;C.β-转角;D.无规则卷曲。

Fig.5 Secondary structure analysis of PlACS protein A.α-helix;B.Extended strand;C.β-turn;D.Random coil.

图6 PlACS蛋白三级结构预测

Fig.6 The tertiary structure prediction of PlACS protein

图7 PlACS基因CDS的扩增及鉴定 A.PlACS基因CDS扩增;B.pMD18-T-PlACS重组质粒PCR; C.pMD18-T-PlACS重组质粒的BamH Ⅰ和Hind Ⅲ双酶切。

Fig.7 PCR amplification and identification of PlACS CDS A.Amplification of PlACS gene CDS;B.PCR identification of pMD18-T-PlACS; C.Double enzyme digestion of pMD18-T-PlACS by BamH Ⅰ/Hind Ⅲ.

图8 PlACS基因原核表达载体pET-32a-PlACS的鉴定 A.PCR;B.BamH Ⅰ和Hind Ⅲ双酶切。

Fig.8 Identification of PlACS prokaryotic expression vector pET-32a-PlACS A.PCR;B.Double enzyme digestion by BamH Ⅰ/Hind Ⅲ.

图9 37 ℃诱导表达的PlACS重组蛋白 M.Protein Marker;1.IPTG未诱导;2.IPTG诱导。

Fig.9 Expression of recombinant PlACS induced at 37 ℃ M.Protein Marker;1.Without induced by IPTG;2.Induced by IPTG.

图10 诱导起始菌体密度对PlACS蛋白表达量的影响 M.Protein Marker;1.未诱导;2—12.A₆₀₀为0.1~1.1时诱导表达。

Fig.10 Effect of bacteria density on expression levels of recombinant PlACS M.Protein Marker;1.Without induced;2—12.Induced when A₆₀₀ was 0.1—1.1.

图11 IPTG 浓度对PlACS蛋白表达量的影响 M.Protein Marker;1.未诱导; 2~10.IPTG终浓度为0.1~0.9 mmol/L。

Fig.11 Effect of IPTG concentration on expression levels of recombinant PlACS M.Protein Marker;1.Without induced; 2—10.The final concentration of IPTG was 0.1-0.9 mmol/L.

图12 诱导时间对PlACS蛋白表达量的影响 M.Protein Marker;1.未诱导;2.2 min;3.5 min;4.10 min;5.20 min;6.40 min;7.1 h;8.2 h;9.4 h。

Fig.12 Effect of induction time on expression levels of recombinant PlACS M.Protein Marker;1.Without induced;2.2 min;3.5 min;4.10 min; 5.20 min;6.40 min;7.1 h;8.2 h;9.4 h.

图13 PlACS 37 ℃表达菌体的裂解上清液及沉淀 M.Protein Marker;1.未诱导上清液;2.诱导上清液;3.未诱导沉淀;4.诱导沉淀。

Fig.13 Lysis supernatant and precipitate of bacteria expressing PlACS at 37 ℃ M.Protein Marker;1.Supernatant without induced; 2.Supernatant induced;3.Precipitate without induced; 4.Precipitate induced.

参考文献 26

[1]	李嘉珏. 中国牡丹与芍药[M]. 北京: 中国林业出版社, 1999.
	Li J J. Chinese tree peony and herbaceous peony[M]. Beijing: China Forestry Publishing House, 1999.
[2]	于晓南, 苑庆磊, 郝丽红. 芍药作为中国爱情花之史考[J]. 北京林业大学学报(社会科学版), 2014, 13(2):26-31.doi: 10.13931/j.cnki.bjfuss.2014.02.005. doi: 10.13931/j.cnki.bjfuss.2014.02.005
	Yu X N, Yuan Q L, Hao L H. Historical analysis of herbaceous peony as symbol of love in China[J]. Journal of Beijing Forestry University (Social Sciences), 2014, 13(2):26-31.
[3]	于晓南. 观赏芍药[M]. 北京: 中国林业出版社, 2019:1-20.
	Yu X N. Ornamental herbaceous peony[M]. Beijing: China Forestry Publishing House, 2019:1-20.
[4]	王哲, 史国安, 马雪情, 范丙友. 芍药桃花飞雪开花衰老期间乙烯代谢生理机制的研究[J]. 园艺学报, 2014, 41(11):2268-2274.doi: 10.16420/j.issn.0513-353x.2014.11.014. doi: 10.16420/j.issn.0513-353x.2014.11.014
	Wang Z, Shi G A, Ma X Q, Fan B Y. The ethylene metabolism in flowers of Chinese peony Taohuafeixue during opening and senescence[J]. Acta Horticulturae Sinica, 2014, 41(11):2268-2274.
[5]	杨若雯, 张萍, 薛玉前, 薛璟祺, 王顺利, 张秀新. 乙烯对班克海尔和杨妃出浴芍药切花开放和衰老的影响[J]. 园艺学报, 2018, 45(8):1575-1586.doi: 10.16420/j.issn.0513-353x.2017-0874. doi: 10.16420/j.issn.0513-353x.2017-0874
	Yang R W, Zhang P, Xue Y Q, Xue J Q, Wang S L, Zhang X X. Effects of ethylene on opening and senescence process of herbaceous peony cut flowers Banker hill and Yangfeichuyu[J]. Acta Horticulturae Sinica, 2018, 45(8):1575-1586.
[6]	Binder B M. Ethylene signaling in plants[J]. Journal of Biological Chemistry, 2020, 295(22):7710-7725.doi: 10.1074/jbc.rev120.010854. doi: 10.1074/jbc.REV120.010854 pmid: 32332098
[7]	Bleecker A B, Kende H. Ethylene:a gaseous signal molecule in plants[J]. Annual Review of Cell and Developmental Biology, 2000, 16:1-18.doi: 10.1146/annurev.cellbio.16.1.1. doi: 10.1146/annurev.cellbio.16.1.1 pmid: 11031228
[8]	Kende H. Enzymes of ethylene biosynthesis[J]. Plant Physiology, 1989, 91(1):1-4.doi: 10.1104/pp.91.1.1. doi: 10.1104/pp.91.1.1 pmid: 16666977
[9]	Yamagami T, Tsuchisaka A, Yamada K, Haddon W F, Harden L A, Theologis A. Biochemical diversity among the 1-amino-cyclopropane-1-carboxylate synthase isozymes encoded by the Arabidopsis gene family[J]. Journal of Biological Chemistry, 2003, 278(49):49102-49112.doi: 10.1074/jbc.m308297200. doi: 10.1074/jbc.M308297200 pmid: 12968022
[10]	Ma N, Cai L, Lu W J, Tan H, Gao J P. Exogenous ethylene influences flower opening of cut roses(Rosa hybrida)by regulating the genes encoding ethylene biosynthesis enzymes[J]. Science in China Series C(Life Sciences), 2005, 48(5):434-444.doi: 10.1360/062004-37. doi: 10.1360/062004-37 URL
[11]	范丙友, 高水平, 刘改秀, 史国安, 李嘉珏, 孔祥生. 牡丹ACS基因片段的克隆及反义植物表达载体构建[J]. 华北农学报, 2010, 25(6):34-37.doi: 10.7668/hbnxb.2010.06.007. doi: 10.7668/hbnxb.2010.06.007
	Fan B Y, Gao S P, Liu G X, Shi G A, Li J J, Kong X S. Cloning of ACC synthase gene from Paeonia suffruticosa and construction of antisense plant expression vector[J]. Acta Agriculturae Boreali-Sinica, 2010, 25(6):34-37.
[12]	高水平, 魏春梅, 刘改秀, 王丽娜, 孔祥生, 范丙友. 牡丹ACS基因克隆及序列分析[J]. 河南农业科学, 2013, 42(3):100-102,106.doi: 10.15933/j.cnki.1004-3268.2013.03.034. doi: 10.15933/j.cnki.1004-3268.2013.03.034
	Gao S P, Wei C M, Liu G X, Wang L N, Kong X S, Fan B Y. Cloning and bioinformatics analysis of gene sequence encoding ACC synthase from Paeonia suffruticosa[J]. Journal of Henan Agricultural Sciences, 2013, 42(3):100-102,106.
[13]	Shi L S, Liu J P. Molecular cloning and expression analysis of an 1-aminocyclopropane-1-carboxylate synthase gene from Oncidium Gower ramsey[J]. Biochemical and Biophysical Research Communications, 2016, 469(2):203-209.doi: 10.1016/j.bbrc.2015.11.107. doi: 10.1016/j.bbrc.2015.11.107 URL
[14]	张帅, 张明, 寇天舒, 马俊莲, 唐霞, 张子德. 无花果ACC合成酶基因克隆及序列分析[J]. 华北农学报, 2012, 27(6):34-37.doi: 10.3969/j.issn.1000-7091.2012.06.008. doi: 10.3969/j.issn.1000-7091.2012.06.008
	Zhang S, Zhang M, Kou T S, Ma J L, Tang X, Zhang Z D. Cloning and sequence analysis of a DNA encoding ACC synthase gene from Ficus carica[J]. Acta Agriculturae Boreali-Sinica, 2012, 27(6):34-37.
[15]	Gasic K, Hernandez A, Korban S S. RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction[J]. Plant Molecular Biology Reporter, 2004, 22(4):437-438.doi: 10.1007/BF02772687. doi: 10.1007/BF02772687 URL
[16]	Xu C, Hao B W, Sun G L, Mei Y Y, Sun L F, Sun Y M, Wang Y B, Zhang Y Y, Zhang W, Zhang M Y, Zhang Y, Wang D, Rao Z H, Li X, Shen Q J, Wang N N. Dual activities of ACC synthase:Novel clues regarding the molecular evolution of ACS genes[J]. Sci Adv, 2021, 7(46):eabg8752.doi: 10.1126/sciadv.abg8752. doi: 10.1126/sciadv.abg8752 URL
[17]	Barry C S, Llop-Tous M I, Grierson D. The regulation of 1-aminocyclopropane-1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato[J]. Plant Physiology, 2000, 123(3):979-986.doi: 10.1104/pp.123.3.979. doi: 10.1104/pp.123.3.979 pmid: 10889246
[18]	Varanasi V, Shin S, Mattheis J, Rudell D, Zhu Y M. Expression profiles of the MdACS3 gene suggest a function as an accelerator of apple(Malus×domestica )fruit ripening[J]. Postharvest Biology and Technology, 2011, 62(2):141-148.doi: 10.1016/j.postharvbio.2011.05.005. doi: 10.1016/j.postharvbio.2011.05.005 URL
[19]	Yip W K, Dong J G, Kenny J W, Thompson G A, Yang S F. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase[J]. Nutrition & Metabolism, 1990, 87(20):7930-7934.doi: 10.1073/pnas.87.20.7930. doi: 10.1073/pnas.87.20.7930
[20]	Lü S Z, Cheng S, Wang Z Y, Li S M, Jin X, Lan L, Yang B, Yu K, Ni X M, Li N, Hou X G, Huang G, Wang J, Dong Y, Wang E Q, Huang J T, Zhang G Y, Zhang C J. Draft genome of the famous ornamental plant Paeonia suffruticosa[J]. Ecology and Evolution, 2020, 10(11):4518-4530.doi: 10.1002/ece3.5965. doi: 10.1002/ece3.5965 URL
[21]	谭远军, 薛璟祺, 仝征, 马男, 高俊平. 切花月季ACC合成酶基因的克隆和原核表达[J]. 园艺学报, 2010, 37(7):1132-1138.doi: 10.16420/j.issn.0513-353x.2010.07.014. doi: 10.16420/j.issn.0513-353x.2010.07.014
	Tan Y J, Xue J Q, Tong Z, Ma N, Gao J P. Cloning and recombinant protein expression of ACC synthase genes in cut roses(Rosa hybrida)[J]. Acta Horticulturae Sinica, 2010, 37(7):1132-1138.
[22]	张超, 张玉环, 贾培义, 董丽. 牡丹ACC合成酶ACS1基因的克隆与原核表达[J]. 生物技术通报, 2011(10):97-100.doi: 10.13560/j.cnki.biotech.bull.1985.2011.10.006. doi: 10.13560/j.cnki.biotech.bull.1985.2011.10.006
	Zhang C, Zhang Y H, Jia P Y, Dong L. Cloning and prokaryotic expression of ACC synthase ACS1 gene from tree peony[J]. Biotechnology Bulletin, 2011(10):97-100.
[23]	高波, 曾庆银, 陆海, 付学奇. 丝瓜ACC合成酶的原核表达、分离纯化及性质鉴定[J]. 吉林大学学报(理学版), 2004, 42(4):622-624.doi: 10.13413/j.cnki.jdxblxb.2004.04.032. doi: 10.13413/j.cnki.jdxblxb.2004.04.032
	Gao B, Zeng Q Y, Lu H, Fu X Q. Prokaryotic expression,purification and activity measurement of towel gourd ACC synthase[J]. Journal of Jilin University (Science Edition), 2004, 42(4):622-624.
[24]	Lilie H, Schwarz E, Rudolph R. Advances in refolding of proteins produced in E.coli[J]. Current Opinion in Biotechnology, 1998, 9(5):497-501.doi: 10.1016/s0958-1669(98)80035-9. doi: 10.1016/s0958-1669(98)80035-9 pmid: 9821278
[25]	Baneyx F. Recombinant protein expression in Escherichia coli[J]. Current Opinion in Biotechnology, 1999, 10(5):411-421.doi: 10.1016/s0958-1669(99)00003-8. doi: 10.1016/s0958-1669(99)00003-8 pmid: 10508629
[26]	范丙友, 胡诗宇, 陆海, 蒋湘宁. 毛白杨4-香豆酸:辅酶A连接酶可溶性原核表达及活性检测[J]. 北京林业大学学报, 2006, 28(2):1-8.doi: 10.3321/j.issn:1000-1522.2006.02.001. doi: 10.3321/j.issn:1000-1522.2006.02.001
	Fan B Y, Hu S Y, Lu H, Jiang X N. Soluble prokaryotic expression of 4-coumarate:Coenzyme A ligase from Populus tomentosa and enzyme activity of recombinant 4CL1[J]. Journal of Beijing Forestry University, 2006, 28(2):1-8.

选择文件类型/文献管理软件名称

选择包含的内容

芍药ACS基因的克隆及其蛋白质原核表达

Cloning and Prokaryotic Expression of ACS Gene from Paeonia lactiflora Pall.

RichHTML

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表 14

参考文献 26

相关文章 15

编辑推荐

Metrics

本文评价

[1]	赵国勇, 高何璇, 刘晓洁, 何玉琴, 陶乐凯, 蔡永强, 张光敏. *甘加型藏羊TACR3基因克隆及在发情周期和乏情期输卵管、子宫中的表达*[J]. 华北农学报, 2025, 40(3): 182-189.
[2]	侯培珂, 程宇坤, 王继庆, 孙玲, 王建鹏, 耿洪伟. *小麦TaSOD-B1基因克隆及KASP标记开发与验证*[J]. 华北农学报, 2025, 40(3): 1-8.
[3]	李忠莹, 郭震楠, 朱洪林, 李佳琳, 施会彬, 韩浩园, 李君, 权凯. *奶山羊SIRT7基因序列特征、组织表达及其对乳脂合成的影响*[J]. 华北农学报, 2025, 40(3): 199-206.
[4]	冯清香, 朱哈修, 杜洋, 徐艳茹, 王晨晨, 刘雪, 李大勇, 周军, 张彬. *菊苣ANS基因的克隆及表达分析*[J]. 华北农学报, 2025, 40(3): 51-59.
[5]	殷冬冬, 朱星星, 兰梦蝶, 彭梦玲, 尹磊, 戴银, 沈学怀, 王洁茹, 赵瑞宏, 潘孝成. 新型鹅星状病毒 AH-2021 株的分离鉴定及其VP27-VP34蛋白多克隆抗体的制备[J]. 华北农学报, 2025, 40(2): 210-217.
[6]	司比努尔·牙生江, 者玉琦, 钟金城, 武志娟, 柴志欣. *九龙牦牛ELOVL3基因克隆、真核表达载体构建及组织表达谱分析*[J]. 华北农学报, 2025, 40(2): 201-209.
[7]	马峻, 连凯琪, 闪金研, 刘玉玲, 李晓龙, 赵黎明, 李学军, 彭仁海. 淇河鲫bmp6基因的克隆及表达特性分析[J]. 华北农学报, 2025, 40(2): 218-229.
[8]	刘永宁, 单乙戈, 潘晨帆, 刘倩琳, 李翌琳, 段思彰, 安健, 张建军. *柔嫩艾美耳球虫AMA1的原核表达及其免疫保护效果评价*[J]. 华北农学报, 2025, 40(1): 232-238.
[9]	黄友举, 于泳波, 逄翠晶, 孙轼絮, 路晨, 于延冲. *大豆GmWRKY44生物信息学分析及功能验证*[J]. 华北农学报, 2025, 40(1): 29-35.
[10]	李俊仁, 陈秀珍, 吴带娣. *广藿香Trihelix家族PatASIL2基因克隆、亚细胞定位及表达分析*[J]. 华北农学报, 2025, 40(1): 104-112.
[11]	彭海芬, 王国君, 段新慧, 刘丽仙, 杨莎莎, 张以芳, 李文贵. 牛流行热病毒云南流行毒株G基因遗传多样性分析[J]. 华北农学报, 2024, 39(S1): 281-288.
[12]	林宏峻, 吕珂, 潘萍萍, 董莉莉, 徐志浩, 王忠华. *金线莲C4H基因克隆与表达及在黄酮合成途径中的作用*[J]. 华北农学报, 2024, 39(S1): 31-38.
[13]	余道宁, 王佟, 洛桑顿珠, 平措占堆, 张强, 卓玛次仁, 尼玛加措, 张德荣, 梁春年. *牦牛PID1基因克隆、生物信息学及组织表达分析*[J]. 华北农学报, 2024, 39(6): 216-223.
[14]	张玉倩, 吕志航, 刘春红, 廉春杨, 张雪莲. 禽戊型肝炎病毒CaHEV-GDSZ01株ORF2蛋白的生物信息学分析、原核表达及多克隆抗体制备[J]. 华北农学报, 2024, 39(6): 231-238.
[15]	赵安琪, 尹跃, 何军, 安巍, 秦小雅, 胡体旭. *枸杞LbaHY5基因克隆、亚细胞定位及表达分析*[J]. 华北农学报, 2024, 39(6): 76-83.

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

芍药ACS基因的克隆及其蛋白质原核表达

Cloning and Prokaryotic Expression of ACS Gene from Paeonia lactiflora Pall.

RichHTML

PDF

可视化

被引次数

摘要/Abstract

引用本文

使用本文

图/表 14

参考文献 26

相关文章 15

编辑推荐

Metrics

本文评价