[1]刘增才,孙婷婷,王世新,等.暴马桑黄MVD基因cDNA全长克隆及表达特性分析[J].南京林业大学学报(自然科学版),2020,44(4):079-85.[doi:10.3969/j.issn.1000-2006.201912007]
 LIU Zengcai,SUN Tingting,WANG Shixin,et al.The cloning and expression analysis of mevalonate pyrophosphate decarboxylase gene cDNA sequence from Sanghuangporus baumii[J].Journal of Nanjing Forestry University(Natural Science Edition),2020,44(4):079-85.[doi:10.3969/j.issn.1000-2006.201912007]
点击复制

暴马桑黄MVD基因cDNA全长克隆及表达特性分析/HTML
分享到:

《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
44
期数:
2020年4期
页码:
079-85
栏目:
研究论文
出版日期:
2020-09-01

文章信息/Info

Title:
The cloning and expression analysis of mevalonate pyrophosphate decarboxylase gene cDNA sequence from Sanghuangporus baumii
文章编号:
1000-2006(2020)04-0079-07
作者:
刘增才 孙婷婷 王世新 马依莎 王旭彤 孙健 邹莉
作者单位:东北林业大学林学院,黑龙江 哈尔滨 150040; 哈尔滨学院食品工程学院,黑龙江 哈尔滨 150086
Author(s):
LIU Zengcai1 SUN Tingting2 WANG Shixin1 MA Yisha1 WANG Xutong1 SUN Jian1 ZOU Li1
(1.College of Forestry, Northeast Forestry University, Harbin 150040, China; 2.Department of Food Engineering, Harbin University, Harbin 150086, China)
关键词:
暴马桑黄 甲羟戊酸焦磷酸脱羧酶 基因克隆 生物信息学分析 表达特性分析
Keywords:
mevalonate pyrophosphate decarboxylase gene cloning bioinformatic analysis expression analysis
分类号:
Q781
DOI:
10.3969/j.issn.1000-2006.201912007
文献标志码:
A
摘要:
目的 对暴马桑黄中参与三萜合成途径的关键酶——甲羟戊酸焦磷酸脱羧酶(MVD)基因进行克隆及表达特性分析,以了解暴马桑黄三萜合成的调控机制。 方法 利用生物信息学软件对MVD基因cDNA序列进行分析,并采取同源重组方法构建原核表达载体。用qRT-PCR技术分析MVD基因在暴马桑黄不同发育阶段表达特性;以分光光度计法测定暴马桑黄在不同发育阶段的三萜含量和变化规律。 结果 暴马桑黄MVD基因cDNA序列全长1 209 bp,编码402个氨基酸,相对分子质量为43.43 ku,命名为 SbMVD(登录号为MK977617)。系统进化树表明:暴马桑黄MVD蛋白和紫芝、灰树花MVD蛋白同源性最高。SDS-PAGE结果显示:在65 ku(包含21 ku标签蛋白)附近出现与预期大小一致的目的蛋白条带。qRT-PCR分析及三萜含量测定结果表明:暴马桑黄SbMVD基因转录水平和三萜含量在不同发育阶段呈先升后降的变化趋势,并且两者变化趋势基本一致。 结论 通过对SbMVD基因的克隆及表达特性分析,推测该基因在三萜合成途径中发挥一定作用,为进一步研究该基因在暴马桑黄三萜合成过程中的功能奠定了基础。
Abstract:
Objective To clone and characterize a mevalonate pyrophosphate decarboxylase (MVD) gene involved in the triterpenoids biosynthesis pathway in Sanghuangporus baumii is to understand the regulation mechanism of the triterpenoids biosynthesis pathway. Method The characteristics of the MVD gene sequence were determined using a series of bioinformatic tools. The prokaryotic expression vector was constructed using homologous recombination. Moreover, qRT-PCR was performed to measure the MVD gene transcript level, and spectrophotometry was used to determine the content and variation of triterpenoids at different developmental stages of S. baumii. Result The sequence analysis showed that the MVD gene cDNA sequence length was 1 209 bp and encoded 402 amino acids. The molecular weight of the protein was predicted to be 43.43 ku, and it was named SbMVD (GenBank number MK977617). Amino acid sequence alignment revealed that the SbMVD protein shared the greatest homology with Ganoderma sinense and Grifola frondosa. SDS-PAGE electrophoresis showed that the target protein band was located at approximately 65 ku (including a 21 ku tag protein), which was consistent with the predicted protein. Furthermore, qRT-PCR and spectrophotometry results showed that the SbMVD gene transcript level and triterpenoids content increased before decreasing dynamically at different developmental stages. The variation trends were basically the same. Conclusion The cloning and analysis of the SbMVD gene indicated that it might play an important role in the triterpenoids biosynthesis pathway. Our results lay a foundation for further elucidating the triterpenoids biosynthesis function of the SbMVD gene in S. baumii.

参考文献/References:

1 Jiangsu New Medical College. Dictionary of chinese medicine[M]. Shanghai: Shanghai Science and Technology Press, 1995.
2 ZHANG W B, WANG J G, LI Z K, et al. Progress of studies on medicinal fungus Phellinus[J]. China J Chin Mater Med, 2014, 39(15): 2838-2845. DOI:10.4268/cjcmm20141510.
3 IKEKAWA T, NAKANISHI M, UEHARA N, et al. Antitumor action of some Basidiomycetes, especially Phellinus linteus[J]. Gann, 1968, 59(2): 155-157. DOI:10.20772/cancersci1959.59.2_155.
4 DING Y Y. Chemical constituents of the medicinal fungus Phellinus igniarius[D]. Hefei: Anhui Medical University, 2017.
5 DAI Y C, ZHOU L W, CUI B K, et al. Current advances in Phellinus sensu lato: medicinal species, functions, metabolites and mechanisms[J]. Appl Microbiol Biotechnol, 2010, 87(5): 1587-1593. DOI:10. 1007/s 00253-010-2711-3.
6 ZHANG L F, SUN T T, ZOU L. Extraction of total triterpenoids from Inonotus baumii and its inhibitory activity on breast cancer cells (MCF-7) in vitro[J]. Drug Eval Res, 2015, 38(5): 497-502. DOI:10.7501/j.issn.1674-6376.2015. 05.006.
7 POTTER D, MIZIORKO H M. Identification of catalytic residues in human mevalonate kinase[J]. J Biol Chem, 1997, 272(41): 25449-25454. DOI:10.1074/jbc.272.41.25449.
8 LANGE B M, CROTEAU R. Isopentenyl diphosphate biosynthesis via a mevalonate?independent pathway: Isopentenyl monophosphate kinase catalyzes the terminal enzymatic step[J]. Proc Natl Acad Sci USA, 1999, 96(24): 13714-13719. DOI:10.1073/pnas.96.24.13714.
9 YANG L, FAN X L, LIU Q, et al. Molecular cloning and analysis of pyrophospomevalonate decarboxylase gene from Paris polyphylla var. Yunnanensis[J]. Chin Tradit Herb Drugs, 2019, 50(6): 1435-1441. DOI: 10.7501/j.issn.0253-2670.2019.06.027.
10 LI Q, LI B, GUO S X. Cloning and expression analysis of pyrophospomevalonate decarboxylase gene in Dendrobium nobile[J]. Genom Appl Biol, 2018, 37(2): 850-858. DOI:10.13417/j.gab.037.000850.
11 SHI L, QIN L, XU Y J, et al. Molecular cloning,characterization, and function analysis of a mevalonate pyrophosphate decarboxylase gene from Ganoderma lucidum[J]. Mol Biol Rep, 2012, 39(5): 6149-6159. DOI:10.1007/s11033-011-1431-9.
12 HOU C X. Cloning of key enzyme MVD gene and βAS gene for ginsenosides biosynthesis and expression research of antisense βAS[D]. Changchun: Jilin University, 2009.
13 XING Z B, LONG Y H, HE S, et al. Cloning and expression analysis of mevalonate diphosphate decarboxylasa gene in Eleutherococcus senticosus[J]. Acta Bot Boreali?Occidentalia Sin, 2012, 32(10): 1950-1956. DOI:10.3969/j.issn.1000-4025.2012.10.003.
14 REDDING?JOHANSON A M, BATTH T S, CHAN R, et al. Targeted proteomics for metabolic pathway optimization: application to terpene production[J]. Metab Eng, 2011, 13(2): 194-203. DOI:10.1016/j.ymben. 2010.12.005.
15 LIAO P, HEMMERLIN A, BACH T J, et al. The potential of the mevalonate pathway for enhanced isoprenoid production[J]. Biotechnol Adv, 2016, 34(5): 697-713. DOI:10.1016/j.biotechadv.2016.03.005.
16 KREPKIY D, MIZIORKO H M. Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases[J]. Protein Sci, 2004, 13(7): 1875-1881. DOI:10.1110/ps.04725204.
17 SUI J J, LI X X, YANG Q Y, et al. Cloning and expression analysis of gene LiSEP3 in double lily[J]. J Nanjing For Univ (Nat Sci Ed), 2017, 41(1): 42-48. DOI:10.3969/j.issn.1000-2006.2017.01. 007.
18 SUN J, SUN T T, WANG X T, et al. Cloning and prokaryotic expression of endoglucanase gene in Auricularia auricular?judae[J]. J Jilin Agric Univ, 2019, 41(3): 308-315. DOI:10.13327/j.jjlau.2019. 4587.
19 SUN T T. Analysis and systematic mining of genes involved in the biosynthetic pathway of triterpenoids in Sanghuangporus baumii[D]. Harbin: Northeast Forestry University, 2017.
20 LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real?time quantitative PCR and the 2-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408. DOI:10.1006/meth.2001.1262.
21 WANG P J, CHEN D, CAO H L, et al. Cloning and expression of mevalonate diphosphate decarboxylase gene CsMVD in tea plant (Camellia sinensis)[J]. Acta Bot Boreali?Occidentalia Sin, 2017, 37(12): 2342-2349. DOI:10.7606/j.issn.1000-4025.2017.12.2342.
22 SUN H P, ZHONG X H, QIAO F. Cloning and expression analysis of mevalonate diphosphate decarboxylase gene in Hedera helix L.[J]. Chin J Trop Crop, 2018, 39(11): 2200-2206. DOI:10.3969/j.issn.1000-2561.2018.11.013.
23 QIN L. Molecular cloning and analysis of expression profiles of mevalonate diphosphate decarboxylase gene from Ganoderma lucidum[D]. Nanjing: Nanjing Agricultural University, 2009.
24 SUN T T, ZOU L, ZHANG L F, et al. Methyl jasmonate induces triterpenoid biosynthesis in Inonotus baumii[J]. Biotechnol Biotechnol Equip, 2017, 31(2): 312-317. DOI:10.1080/13102818.2017.1284023.
25 XU Y N, XIA X X, ZHONG J J. Induction of ganoderic acid biosynthesis by Mn2+ in static liquid cultivation of Ganoderma lucidum[J]. Biotechnol Bioeng, 2014, 111(11): 2358-2365. DOI:10.1002/bit.25288.

备注/Memo

备注/Memo:
收稿日期:2019-12-04
更新日期/Last Update: 2020-08-13