药学学报, 2019, 54(8): 1515-1523
引用本文:
刘露, 周良云, 张春荣, 王浩, 刘长征, 杨全. 何首乌中UDP-鼠李糖合成酶基因FmRHM1/2的克隆与鉴定[J]. 药学学报, 2019, 54(8): 1515-1523.
LIU Lu, ZHOU Liang-yun, ZHANG Chun-rong, WANG Hao, LIU Chang-zheng, YANG Quan. Cloning and characterization of UDP-L-rhamnose synthase 1/2 from Fallopia multiflora[J]. Acta Pharmaceutica Sinica, 2019, 54(8): 1515-1523.

何首乌中UDP-鼠李糖合成酶基因FmRHM1/2的克隆与鉴定
刘露, 周良云, 张春荣, 王浩, 刘长征, 杨全
广东药科大学中药学院/国家中医药管理局岭南药材生产与开发重点研究室/国家中药材产业技术体系广州综合试验站/广东省南药规范化种植与综合开发工程技术研究中心, 广东 广州 510006
摘要:
UDP-鼠李糖是一种由UDP-鼠李糖合酶(RHM)催化合成的鼠李糖供体,而鼠李糖是鼠李糖苷化合物的重要组成部分,植物中只有少数基因编码的酶参与UDP-鼠李糖生物合成。本研究基于何首乌(Fallopia multiflora(Thunb.) Harald.)转录组数据,首次克隆得到2个RHM基因(FmRHM1FmRHM2),并进行生物学信息分析、体外功能鉴定及组织特异性分析。结果显示FmRHM1/2基因的开放阅读框均为2013 bp,均编码670个氨基酸,推测蛋白质分子质量均为75.6 kDa,理论等电点分别为6.20和7.19,具有RHM酶家族的特征信号序列(GxxGxxG/A和YxxxK);多序列比对与系统进化树显示,FmRHM与其他物种的RHM具有同源性。体外酶促反应结果显示,重组蛋白FmRHM1和FmRHM2均具有催化活性,可将UDP-葡萄糖转化为UDP-鼠李糖。组织特异性表达显示,FmRHM1FmRHM2基因在根中的表达量最低,并与茎和叶相比均存在显著性差异。本研究首次报道了何首乌RHM,并验证了其催化功能,为进一步研究微生物合成UDP-鼠李糖奠定基础。
关键词:    何首乌      UDP-L-鼠李糖      UDP-鼠李糖合成酶      功能鉴定     
Cloning and characterization of UDP-L-rhamnose synthase 1/2 from Fallopia multiflora
LIU Lu, ZHOU Liang-yun, ZHANG Chun-rong, WANG Hao, LIU Chang-zheng, YANG Quan
Key Laboratory of State Administration of Traditional Chinese Medicine for Production and Development of Cantonese Medicinal Materials, Guangzhou Comprehensive Experimental Station of National Industrial Technology System for Chinese Materia Medica, Guangdong Engineering Research Center of Good Agricultural Practice and Comprehensive Development for Cantonese Medicinal Materials, School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China
Abstract:
UDP-rhamnose is a rhamnose donor in a reaction catalyzed by UDP-rhamnose synthase (RHM), and plays an important role in the biosynthesis of rhamnoside compounds. The current literature suggests that there are only a few genes can encode the corresponding enzymes to participate in UDP-rhamnose biosynthesis in plants. In this study, two RHM genes (FmRHM1 & 2) were first cloned by using the transcriptomic data of Fallopia multiflora (Thunb) Harald and the multidimensional analysis, including bioinformatics, functional identification in vitro and tissue-specific expression analysis. The results showed that the open reading frame (ORF) of FmRHM1 & 2 genes both were 2 013 bp, encode proteins consisting of 670 amino acids with a calculated molecular mass of 75.6 kDa, and the theoretical isoelectric points of 6.20 and 7.19, respectively. Bioinformatic analysis also indicated that FmRHM1 & 2 contained 2 special sequences of GxxGxxG/A and YxxxK. The phylogenetic analysis showed that the FmRHM gene has a high homology with RHM of other species. The results of enzyme activity in vitro revealed that both recombinant FmRHM1 and FmRHM2 have catalytic activities for converting UDP-glucose into UDP-rhamnose. Measurements of tissue-specific expressions showed that the expression levels of FmRHM1 and FmRHM2 were lower in roots. On the contrary, the 2 genes showed significantly high expression in the stems and leaves. In conclusion, we have cloned and characterized the RHM gene function for the first time in F. multiflora. Here we have provided the preliminary data suggesting the need for further research on UDP-rhamnose biosynthesis by microorganisms.
Key words:    Fallopia multiflora    UDP-rhamnose    UDP rhamnose synthase    function characterization   
收稿日期: 2019-03-19
DOI: 10.16438/j.0513-4870.2019-0184
基金项目: 国家重点研发计划项目(2017YFC1700704);2017年广东省岭南中药材保护资金专项(粤财社[2017]60号).
通讯作者: 杨全,Tel/Fax:86-20-39352353,E-mail:yangquan7208@vip.163.com
Email: yangquan7208@vip.163.com
相关功能
PDF(1249KB) Free
打印本文
0
作者相关文章
刘露  在本刊中的所有文章
周良云  在本刊中的所有文章
张春荣  在本刊中的所有文章
王浩  在本刊中的所有文章
刘长征  在本刊中的所有文章
杨全  在本刊中的所有文章

参考文献:
[1] Kim BG, Kim HJ, Ahn JH. Production of bioactive flavonol rhamnosides by expression of plant genes in Escherichia coli[J]. J Agric Food Chem, 2012, 60:11143-11148.
[2] Oka T, Nemoto T, Jigami Y. Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion[J]. J Biol Chem, 2007, 282:5389-5403.
[3] Manzanares P, Vallés S, Ramòn D, et al. α-L-Rhamnosidases:old and new insights//Polaina J, MacCabe AP (eds) Industrial Enzymes[M]. Dordrecht:Springer, 2007:117-140.
[4] Rho HS, Ghimeray AK, Yoo DS, et al. Kaempferol and kaempferol rhamnosides with depigmenting and anti-inflammatory properties[J]. Molecules, 2011, 16:3338-3344.
[5] Choi HJ, Kim JH, Lee CH, et al. Antiviral activity of quercetin 7-rhamnoside against porcine epidemic diarrhea virus[J]. Antiviral Res, 2009, 81:77-81.
[6] Hayder N, Bouhlel I, Skandrani I, et al. In vitro antioxidant and antigenotoxic potentials of myricetin-3-O-galactoside and myricetin-3-O-rhamnoside from Myrtus communis:modulation of expression of genes involved in cell defence system using cDNA microarray[J]. Toxicol in Vitro, 2008, 22:567-581.
[7] Diantini A, Subarnas A, Lestari K, et al. Kaempferol-3-Orhamnoside isolated from the leaves of Schima wallichii Korth. inhibits MCF-7 breast cancer cell proliferation through activation of the caspase cascade pathway[J]. Oncol Lett, 2012, 3:1069-1072.
[8] Kamsteeg J, Brederode JV, Nigtevecht GV. The formation of UDP-L-rhamnose from UDP-D-glucose by an enzyme preparation of red campion (Silene dioica (L) Clairv) leaves[J]. FEBS Lett, 1978, 91:281-284.
[9] Jiang XM, Neal B, Santiago F, et al. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2)[J]. Mol Microbiol, 2010, 5:695-713.
[10] Yoo HG, Kwon SY, Karki S, et al. A new route to dTDP-6-deoxy-L-talose and dTDP-L-rhamnose:dTDP-L-rhamnose 4-epimerase in Burkholderia thailandensis[J]. Bioorg Med Chem Lett, 2011, 21:3914-3917.
[11] Giraud MF, Leonard GA, Field RA, et al. RmlC, the third enzyme of dTDP-L-rhamnose pathway, is a new class of epimerase[J]. Nat Struct Biol, 2000, 7:398-402.
[12] Blankenfeldt W, Kerr ID, Giraud MF, et al. Variation on a theme of SDR:dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) shows a new Mg2+-dependent dimerization mode[J]. Structure, 2002, 10:773-786.
[13] Martinez V, Ingwers M, Smith J, et al. Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi Magnaporthe grisea and Botryotinia fuckeliana[J]. J Biol Chem, 2012, 287:879-892.
[14] Zhao Y, Thorson J. A methodological comparison:the advantage of phosphorimidates in expanding the sugar nucleotide repertoire[J]. J Org Chem, 1998, 63:7568-7572.
[15] Sun Q, Li XJ, Sun J, et al. An improved P (V)-N activation strategy for the synthesis of nucleoside diphosphate 6-deoxy-Lsugars[J]. Tetrahedron, 2014, 70:294-300.
[16] Kim B, Jung W, Ahn J. Cloning and characterization of a putative UDP-rhamnose synthase 1 from Populus Euramericana Guinier[J]. J Plant Biol, 2013, 56:7-12.
[17] Casas MI, Falcone-Ferreyra ML, Jiang N, et al. Identification and characterization of maize salmon silks genes involved in insecticidal maysin biosynthesis[J]. Plant Cell, 2016, 28:1297-1309.
[18] Zhao MY. Chemical constituents and pharmacology from the root of Polygonum multiflorum[J]. J North Pharm (北方药学), 2018, 15:192-193.
[19] Yuan W, Gao ZP, Yang JB, et al. Chemical constituents from Polygonum multiflorum[J]. Chin Tradit Herb Drugs (中草药), 2017, 48:631-634.
[20] Han X, Qian L, Zhang L, et al. Structural and biochemical insights into nucleotide-rhamnose synthase/epimerase-reductase from Arabidopsis thaliana[J]. Biochim Biophys Acta, 2015, 1854:1476-1486.
[21] Han XD. Structural Studies of L-Sorbose Dehydrogenase and Nucleotide-Rhamnose Synthetase (山梨糖脱氢酶与鼠李糖合成酶结构与功能的研究)[D]. Tianjin:Nankai University, 2014.
[22] Eixelsberger T, Nidetzky B. Enzymatic redox cascade for onepot synthesis of uridine 5'-diphosphate xylose from uridine 5'-diphosphate glucose[J]. Adv Synth Catal, 2015, 356:3575-3584.