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

doi:10.7668/hbnxb.20193096

Abstract

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

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.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20193096

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

Figures/Tables 14

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

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

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.

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

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

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

Fig.6 The tertiary structure prediction of PlACS protein

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.

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

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

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.

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.

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.

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.

References 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.

Please choose a citation manager

Content to export