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.

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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.

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