紫苏甲羟戊酸-5-磷酸激酶基因<i>PfPMK</i>的克隆与表达分析

doi:10.7668/hbnxb.20192609

摘要/Abstract

摘要：

为了研究甲羟戊酸-5-磷酸激酶(PMK)在紫苏萜类物质生物合成代谢通路中的重要作用,对紫苏转录组数据进行分析,挖掘出紫苏PMK基因参考序列,采用基因克隆技术从紫苏中克隆了PMK基因,利用生物信息学和实时荧光定量PCR(qRT-PCR)的方法对紫苏PMK基因进行了分析。结果表明,紫苏PMK基因开放阅读框(ORF)全长为1 524 bp,编码507个氨基酸。生物信息学分析显示,PfPMK的分子质量为54.73 ku,等电点为5.20,为亲水蛋白;紫苏PfPMK氨基酸与SmPMK、SsPMK、SbPMK和PvPMK同源性较高,说明紫苏PfPMK蛋白在进化过程中保守性较强。亲缘关系分析显示,PfPMK与丹参和一串红的PMK亲缘关系很近,在线软件WoLF-PSORT预测PfPMK蛋白可能定位于质膜或内质网膜上。qRT-PCR结果表明,PfPMK基因在紫苏根、茎、叶中均有表达,在根中的表达量高于在叶和茎中的表达量;紫苏不同生长发育时期的qRT-PCR分析显示,PfPMK基因在9月中下旬表达量较高。首次从紫苏中克隆出PfPMK基因,生物信息学分析该基因属于甲羟戊酸-5-磷酸激酶基因,参与了紫苏萜类物质的生物合成。

关键词: 紫苏, PfPMK, 萜类, 生物信息学, 功能分析

Abstract:

In order to investigate the function of 5-phosphomevalonate kinase PMK gene in biosynthesis pathway of terpenoids in Perilla frutescens L.,we analyzed the transcriptome data of Perilla frutescens and mined the reference sequence of PMK.The PMK gene was cloned from Perilla frutescens by gene cloning technique,and was analysed using bioinformatics and Real-time PCR(qRT-PCR).The results showed that the ORF of PfPMK gene was 1 524 bp,encoding 507 amino acids.Bioinformatics analysis showed that the molecular weight of PfPMK was 54.73 ku and the isoelectric point was 5.20,which was a hydrophilic protein.The amino acids of PfPMK were higher homologous with SmPMK,SsPMK,SbPMK and PvPMK,which indicated PfPMK protein was highly conserved in the evolutionary process.The phylogenetic analysis showed that PfPMK was closely related to PMK of Salvia miltiorrhiza and Salvia splendens.WoLF-PSORT predicted that PfPMK protein might be located in the plasma membrane or endoplasmic reticulum.Real-time fluorescence quantitative PCR results showed that PfPMK gene was expressed in roots,stems and leaves of Perilla frutescens,and the expression level in roots was higher than that in leaves and stems.The fluorescence quantitative PCR analysis showed that the expression level of PfPMK gene was higher in the middle and late September.The full length of PfPMK gene was first cloned from Perilla frutescens.Bioinformatics analysis showed that PfPMK gene belonged to 5-phosphomevalonate kinase gene and participates in the biosynthesis of terpenoids in Perilla frutescens.

Key words: Perilla frutescens L., PfPMK, Terpenoids, Bioinformatics, Functional analysis

李辉, 温春秀, 刘灵娣, 温赛群, 唐映红, 姜涛. 紫苏甲羟戊酸-5-磷酸激酶基因PfPMK的克隆与表达分析[J]. 华北农学报, 2022, 37(2): 49-55. doi: 10.7668/hbnxb.20192609.

LI Hui, WEN Chunxiu, LIU Lingdi, WEN Saiqun, TANG Yinghong, JIANG Tao. Cloning and Expression Analysis of 5-Phosphomevalonate Kinase PfPMK Gene in Perilla frutescens L.[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 49-55. doi: 10.7668/hbnxb.20192609.

http://www.hbnxb.net/CN/Y2022/V37/I2/49

图/表 5

图1 PfPMK基因克隆及氨基酸序列

Fig.1 Cloning of PfPMK gene and amino acid sequence

图2 PfPMK基因生物信息学分析

Fig.2 Bioinformatics analysis of PfPMK gene

图3 PfPMK蛋白分析

Fig.3 Protein analysis of PfPMK

图4 PfPMK氨基酸结构预测

Fig.4 Amino acid structure prediction of PfPMK

图5 PfPMK基因表达分析
不同小写字母表示与对照相比差异显著(P<0.05)。

Fig.5 Expression analysis of PfPMK gene
Different lowercase letters indicate significant difference compared with the control(P<0.05).

参考文献 28

[1]	Jiang T,Guo K Y,Liu L D,Tian W,Xie X L,Wen S Q,Wen C X.Integrated transcriptomic and metabolomic data reveal the flavonoid biosynthesis metabolic pathway in Perilla frutescens(L.)leaves[J].Scientific Reports,2020,10:16207.doi:10.1038/s41598-020-73274-y.
[2]	胡彦. 中国紫苏属植物种源评价及紫苏多倍体育种的初步研究[D].北京:北京林业大学,2010.
	Hu Y.Preliminary studies on the evaluation resources and polyploid breeding of Perilla L.in China[D].Beijing:Beijing Forestry University,2010.
[3]	Liao Z H,Chen M,Gong Y F,Miao Z Q,Sun X F,Tang K X.Isoprenoid biosynthesis in plants:Pathways,genes,regulation and metabolic engineering[J].Journal of Biological Sciences,2005,6(1):209-219.doi:10.3923/jbs.2006.209.219.
[4]	Sando T,Takaoka C,Mukai Y,Yamashita A,Hattori M,Ogasawara N,Fukusaki E,Kobayashi A.Cloning and characterization of mevalonate pathway genes in a natural rubber producing plant,Hevea brasiliensis[J].Bioscience,Biotechnology,and Biochemistry,2008,72(8):2049-2060.doi:10.1271/bbb.80165.
[5]	Niu M Y,Xiong Y P,Yan H F,Zhang X H,Li Y,da Silva J A T,Ma G H.Cloning and expression analysis of mevalonate kinase and phosphomevalonate kinase genes associated with the MVA pathway in Santalum album[J].Scientific Reports,2021,11(1):16913.doi:10.1038/s41598-021-96511-4.
[6]	Xiao W J,Chang H P,Zhou P,Yuan C Y,Zhang C,Yao R Y,Lu X T,Cai L J,He Z,Hu R,Guo X H.Genome-wide identification,classification and expression analysis of GHMP genes family in Arabidopsis thaliana[J].Plant Systematics and Evolution,2015,301(8):2125-2140.doi:10.1007/s00606-015-1219-9.
[7]	Ma Y M,Yuan L C,Wu B,Li X E,Chen S L,Lu S F.Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza[J].Journal of Experimental Botany,2012,63(7):2809-2823.doi:10.1093/jxb/err466.
[8]	杨恩泽,陆颖锶,吴秋红,王峰.阳春砂磷酸甲羟戊酸激酶的基因克隆与表达谱分析[J].暨南大学学报(自然科学与医学版),2015,36(2):95-101.doi:10.11778/j.jdxb.2015.02.001.
	Yang E Z,Lu Y S,Wu Q H,Wang F.Cloning and expression profiles analysis of PMK gene in Amomum villosum Lour[J].Journal of Jinan University (Natural Science & Medicine Edition),2015,36(2):95-101.
[9]	陈笛,王鹏杰,郑玉成,郑知临,陈桂信,叶乃兴.茉莉花磷酸甲羟戊酸激酶基因克隆及表达分析[J].南方农业学报,2018,49(10):1909-1916.doi:10.3969/j.issn.2095-1191.2018.10.02.
	Chen D,Wang P J,Zheng Y C,Zheng Z L,Chen G X,Ye N X.Cloning and expression analysis of phosphomevalonate kinase gene from Jasminum sambac(L.)Ait[J].Journal of Southern Agriculture,2018,49(10):1909-1916.
[10]	赵乐,马利刚,杨方方,冯卫生,郑晓珂.独行菜磷酸甲羟戊酸激酶LaPMK基因克隆、生物信息学分析及原核表达[J].中草药,2016,47(17):3087-3093.doi:10.7501/j.issn.0253-2670.2016.17.021.
	Zhao L,Ma L G,Yang F F,Feng W S,Zheng X K.Cloning,bioinformatic analysis,and prokaryotic expression of LaPMK gene in Lepidium apetalum[J].Chinese Traditional and Herbal Drugs,2016,47(17):3087-3093.
[11]	郑汉,虞慕瑶,濮春娟,陈美兰,李福全,申业,黄璐琦.香樟甲羟戊酸-5-磷酸激酶基因CcPMK的克隆和表达分析[J].中国中药杂志,2020,45(1):78-84.doi:10.19540/j.cnki.cjcmm.20191104.104.
	Zheng H,Yu M Y,Pu C J,Chen M L,Li F Q,Shen Y,Huang L Q.Cloning and expression analysis of 5-phosphomevalonate kinase gene(CcPMK)in Cinnamomum camphora[J].China Journal of Chinese Materia Medica,2020,45(1):78-84.
[12]	姜涛,刘灵娣,田伟,谢晓亮,温春秀.紫苏SNP分子标记开发及遗传多样性分析[J].分子植物育种,2021,19(4):1243-1249.doi:10.13271/j.mpb.019.001243.
	Jiang T,Liu L D,Tian W,Xie X L,Wen C X.SNP molecular markers development and genetic diversity analysis of Perilla frutescens(L.)[J].Molecular Plant Breeding,2021,19(4):1243-1249.
[13]	国家药典委员会.中华人民共和国药典[M].北京:中国医学科学出版社,2020.
	National Pharmacopoeia Commission.The People's Republic of China pharmacopoeia[M].Beijing:China Medical Science Press,2020.
[14]	Luo Y J,Guo J Q,Li W P,Yao Y,Wen C M,Guo B L.Contents determination of eight phenolic compounds in Perilla frutescens leaves of different cultivation years and harvesting periods[J].China Journal of Chinese Materia Medica,2021,46(3):567-574.doi:10.19540/j.cnki.cjcmm.20201114.101.
[15]	Ju H J,Bang J H,Chung J W,Hyun T K.Variation in essential oil composition and antimicrobial activity among different genotypes of Perilla frutescens var.crispa[J].Journal of Applied Biological Chemistry,2021,64(2):127-131.doi:10.3839/jabc.2021.019.
[16]	Paradee N,Utama-Ang N,Uthaipibull C,Porter J B,Garbowski M W,Srichairatanakool S.Extracts of Thai Perilla frutescens nutlets attenuate tumour necrosis factor-α-activated generation of microparticles, ICAM-1 and IL-6 in human endothelial cells[J].Bioscience Reports,2020,40(5):BSR20192110.doi:10.1042/bsr20192110.
[17]	You C X,Yang K,Wu Y,Zhang W J,Wang Y,Geng Z F,Chen H P,Jiang H Y,Du S S,Deng Z W,Liu Z L.Chemical composition and insecticidal activities of the essential oil of Perilla frutescens(L.)Britt.aerial parts against two stored product insects[J].European Food Research and Technology,2014,239(3):481-490.doi:10.1007/s00217-014-2242-8.
[18]	Masumoto N,Nishizaki Y,Maruyama T,Igarashi Y,Nakajima K,Yamazaki T,Kuroe M,Numata M,Ihara T,Sugimoto N,Sato K.Determination of perillaldehyde in Perilla herbs using relative molar sensitivity to single-reference diphenyl sulfone[J].Journal of Natural Medicines,2019,73(3):566-576.doi:10.1007/s11418-019-01306-7.
[19]	Jones C G,Plummer J A,Barbour E L.Non-destructive sampling of Indian sandalwood(Santalum album L.)for oil content and composition[J].Journal of Essential Oil Research,2007,19(2):157-164.doi:10.1080/10412905.2007.9699250.
[20]	Jones C G,Keeling C I,Ghisalberti E L,Barbour E L,Plummer J A,Bohlmann J.Isolation of cDNAs and functional characterisation of two multi-product terpene synthase enzymes from sandalwood,Santalum album L.[J].Archives of Biochemistry and Biophysics,2008,477(1):121-130.doi:10.1016/j.abb.2008.05.008.
[21]	Rani A,Ravikumar P,Reddy M D,Kush A.Molecular regulation of santalol biosynthesis in Santalum album L[J].Gene,2013,527(2):642-648.doi:10.1016/j.gene.2013.06.080.
[22]	Zhang X H,Niu M Y,Teixeira da Silva J A,Zhang Y Y,Yuan Y F,Jia Y X,Xiao Y Y,Li Y,Fang L,Zeng S J,Ma G H.Identification and functional characterization of three new terpene synthase genes involved in chemical defense and abiotic stresses in Santalum album[J].BMC Plant Biology,2019,19(1):115.doi:10.1186/s12870-019-1720-3.
[23]	Diaz-Chavez M L,Moniodis J,Madilao L L,Jancsik S,Keeling C I,Barbour E L,Ghisalberti E L,Plummer J A,Jones C G,Bohlmann J.Biosynthesis of sandalwood oil:Santalum album CYP76F cytochromes P450 produce santalols and bergamotol[J].PLoS One,2013,8(9):e75053.doi:10.1371/journal.pone.0075053.
[24]	Celedon J M,Chiang A,Yuen M M S,Diaz-Chavez M L,Madilao L L,Finnegan P M,Barbour E L,Bohlmann J.Heartwood-specific transcriptome and metabolite signatures of tropical sandalwood(Santalum album)reveal the final step of(Z)-santalol fragrance biosynthesis[J].The Plant Journal,2016,86(4):289-299.doi:10.1111/tpj.13162.
[25]	Song Q L,Meng X X,Liao Y L,Zhang W W,Ye J B,Xu F.Transcriptome-guided gene isolation,characterization and expression analysis of a phosphomevalonate kinase gene(GbPMK)from Ginkgo biloba[J].International Journal of Agriculture and Biology,2018,20(5):1080-1088.doi:10.17957/IJAB/15.0612.
[26]	Woo H M,Murray G W,Batth T S,Prasad N,Adams P D,Keasling J D,Petzold C J,Lee T S.Application of targeted proteomics and biological parts assembly in E.coli to optimize the biosynthesis of an anti-malarial drug precursor,Amorpha-4,11-diene[J].Chemical Engineering Science,2013,103:21-28.doi:10.1016/j.ces.2013.04.033.
[27]	Simkin A J,Guirimand G,Papon N,Courdavault V,Thabet I,Ginis O,Bouzid S,Giglioli-Guivarc'h N,Clastre M.Peroxisomal localisation of the final steps of the mevalonic acid pathway in planta[J].Planta,2011,234(5):903-914.doi:10.1007/s00425-011-1444-6.
[28]	Kim Y K,Kim Y B,Kim J K,Kim S U,Park S U.Molecular cloning and characterization of mevalonic acid(MVA)pathway genes and triterpene accumulation in Panax ginseng[J].Journal of the Korean Society for Applied Biological Chemistry,2014,57(3):289-295.doi:10.1007/s13765-014-4008-1.

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紫苏甲羟戊酸-5-磷酸激酶基因PfPMK的克隆与表达分析

Cloning and Expression Analysis of 5-Phosphomevalonate Kinase PfPMK Gene in Perilla frutescens L.

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紫苏甲羟戊酸-5-磷酸激酶基因PfPMK的克隆与表达分析

Cloning and Expression Analysis of 5-Phosphomevalonate Kinase PfPMK Gene in Perilla frutescens L.

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