暴马桑黄MVD基因cDNA全长克隆及表达特性分析

doi:10.3969/j.issn.1000-2006.201912007

国家林草科技领军期刊
中国精品科技期刊
中国高校百佳科技期刊
江苏省新闻出版政府奖期刊奖
RCCSE林学权威期刊（A+）
CSCD核心期刊
Scopus数据库收录期刊
中文核心期刊
SCD核心期刊

作者加群：102861116

微信公众号：南京林业大学学报

高级检索

南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (4): 79-85.doi: 10.3969/j.issn.1000-2006.201912007

暴马桑黄MVD基因cDNA全长克隆及表达特性分析

刘增才¹(), 孙婷婷², 王世新¹, 马依莎¹, 王旭彤¹, 孙健¹, 邹莉¹()

^1.东北林业大学林学院，黑龙江哈尔滨 150040
^2.哈尔滨学院食品工程学院，黑龙江哈尔滨 150086

收稿日期:2019-12-04 修回日期:2020-02-27 出版日期:2020-07-22 发布日期:2020-08-13
通讯作者: 邹莉
作者简介:刘增才（1758458181@qq.com）
基金资助:
中央高校基本科研业务费专项资金资助项目(2572018AA34)

The cloning and expression analysis of mevalonate pyrophosphate decarboxylase gene cDNA sequence from Sanghuangporus baumii

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

Received:2019-12-04 Revised:2020-02-27 Online:2020-07-22 Published:2020-08-13
Contact: ZOU Li

摘要/Abstract

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。

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

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.

Key words: Sanghuangporus baumii, mevalonate pyrophosphate decarboxylase, gene cloning, bioinformatic analysis, expression analysis

中图分类号:

Q781

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): 79-85.

LIU Zengcai, SUN Tingting, WANG Shixin, MA Yisha, WANG Xutong, SUN Jian, ZOU Li. 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): 79-85.DOI: 10.3969/j.issn.1000-2006.201912007.

图/表 8

表1

图1

图2

图3

图4

图5

图6

图7

参考文献 25

1	江苏新医学院.中药大辞典[M]. 上海: 上海科学技术出版社, 1995.
	Jiangsu New Medical College. Dictionary of chinese medicine[M]. Shanghai: Shanghai Science and Technology Press, 1995.
2	张维博, 王家国, 李正阔, 等. 药用真菌桑黄的研究进展[J]. 中国中药杂志, 2014, 39(15): 2838-2845.
	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	丁云云. 药用真菌桑黄化学成分的研究[D]. 合肥: 安徽医科大学, 2017.
	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	张林芳, 孙婷婷, 邹莉. 鲍姆纤孔菌总三萜的提取及其体外抗乳腺癌细胞MCF-7活性[J]. 药物评价研究, 2015, 38(5): 497-502.
	ZHANG L F, SUN T T, ZOU L. Extraction of total triterpenoids from Inonotusbaumii 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	杨莉, 樊小莉, 刘琴, 等. 滇重楼甲羟戊酸焦磷酸脱羧酶基因的分子克隆与分析[J]. 中草药, 2019, 50(6): 1435-1441.
	YANG L, FAN X L, LIU Q, et al. Molecular cloning and analysis of pyrophospomevalonate decarboxylase gene from Parispolyphylla var. Yunnanensis[J]. Chin Tradit Herb Drugs, 2019, 50(6): 1435-1441. DOI: 10.7501/j.issn.0253-2670.2019.06.027.
10	李清, 李标, 郭顺星. 金钗石斛焦磷酸甲羟戊酸脱羧酶基因的克隆及表达分析[J]. 基因组学与应用生物学, 2018, 37(2): 850-858.
	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 Ganodermalucidum[J]. Mol Biol Rep, 2012, 39(5): 6149-6159. DOI:10.1007/s11033-011-1431-9.
12	侯春喜. 人参皂苷生物合成关键酶基因MVD和βAS的克隆及βAS的反义表达[D]. 长春: 吉林大学, 2009.
	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	邢朝斌, 龙月红, 何闪, 等. 刺五加甲羟戊酸焦磷酸脱羧酶基因的克隆与表达分析[J]. 西北植物学报, 2012, 32(10): 1950-1956.
	XING Z B, LONG Y H, HE S, et al. Cloning and expression analysis of mevalonate diphosphate decarboxylasa gene in Eleutherococcussenticosus[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	隋娟娟, 李晓昕, 杨秋燕, 等. 重瓣百合LiSEP3基因克隆与表达分析[J]. 南京林业大学学报(自然科学版), 2017, 41(1): 42-48.
	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	孙健, 孙婷婷, 王旭彤, 等. 黑木耳内切葡聚糖酶基因克隆与原核表达[J]. 吉林农业大学学报, 2019, 41(3): 308-315.
	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	孙婷婷. 桑黄三萜生物合成途径关键酶基因的挖掘及分析[D]. 哈尔滨: 东北林业大学, 2017.
	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	王鹏杰, 陈丹, 曹红利, 等. 茶树甲羟戊酸焦磷酸脱羧酶基因CsMVD的克隆与表达分析[J]. 西北植物学报, 2017, 37(12): 2342-2349.
	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	孙化鹏, 钟晓红, 乔飞. 洋常春藤甲羟戊酸焦磷酸脱羧酶基因HhMVD的克隆与表达分析[J]. 热带作物学报, 2018, 39(11): 2200-2206.
	SUN H P, ZHONG X H, QIAO F. Cloning and expression analysis of mevalonate diphosphate decarboxylase gene in Hederahelix L.[J]. Chin J Trop Crop, 2018, 39(11): 2200-2206. DOI:10.3969/j.issn.1000-2561.2018.11.013.
23	秦磊. 灵芝甲羟戊酸焦磷酸脱羧酶基因的克隆及表达特性研究[D]. 南京: 南京农业大学, 2009.
	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.

[1]	刘增才, 王淑婷, 佟鑫宇, 邹莉. 暴马桑黄GPS基因克隆及响应茉莉酸甲酯诱导表达研究[J]. 南京林业大学学报（自然科学版）, 2023, 47(4): 123-130.
[2]	魏祯祯, 宋程威, 郭丽丽, 郭琪, 侯小改. ‘凤丹’PoERF4基因的克隆及表达分析[J]. 南京林业大学学报（自然科学版）, 2023, 47(3): 56-62.
[3]	贾展慧, 贾晓东, 许梦洋, 莫正海, 翟敏, 宣继萍, 张计育, 王刚, 王涛, 郭忠仁. 薄壳山核桃原花青素合成关键酶基因的克隆与表达分析[J]. 南京林业大学学报（自然科学版）, 2022, 46(5): 49-57.
[4]	林莉莉, 胡安琪, 陈钢, 张霁月, 曹光球, 曹世江. 杉木ClWRKY44基因克隆及其表达特性分析[J]. 南京林业大学学报（自然科学版）, 2022, 46(1): 203-209.
[5]	黎梦娟, 朱礼明, 霍俊男, 张景波, 施季森, 成铁龙. 唐古特白刺NtCBL1、NtCBL2基因克隆及表达分析[J]. 南京林业大学学报（自然科学版）, 2021, 45(3): 93-99.
[6]	张腾倩, 张伟溪, 丁昌俊, 张静, 胡赞民, 苏晓华. 欧美杨PdMODD基因克隆与表达特性分析[J]. 南京林业大学学报（自然科学版）, 2021, 45(2): 43-50.
[7]	张雨, 丰凯, 汤方. 杨小舟蛾MtroOBP1基因的克隆、序列分析及表达[J]. 南京林业大学学报（自然科学版）, 2020, 44(5): 215-221.
[8]	周晓峰, 莫正海, 尚杨娟, 骆敏, 苏文川, 谭鹏鹏, 韩明慧, 邓秋菊, 田朝霞, 彭方仁. 薄壳山核桃CiDGAT1基因的克隆及表达模式分析[J]. 南京林业大学学报（自然科学版）, 2020, 44(4): 55-62.
[9]	孙晓波, 陈佩珍, 吴晓刚, 吴帆, 季孔庶. 马尾松PmAOX基因克隆与不同逆境胁迫表达分析[J]. 南京林业大学学报（自然科学版）, 2020, 44(4): 70-78.
[10]	刘玉梅, 赵焕元, 崔茂凯, 王田雨, 高向倩, 杨桂燕. 核桃JrERF2⁃2基因克隆及其对逆境的响应[J]. 南京林业大学学报（自然科学版）, 2020, 44(3): 58-64.
[11]	田雪瑶, 周洁, 王保松, 何开跃, 何旭东. 柳树NAC基因的克隆与表达模式分析[J]. 南京林业大学学报（自然科学版）, 2020, 44(1): 119-124.
[12]	莫正海, 李风达, 苏文川, 曹凡, 彭方仁, 李永荣. 薄壳山核桃嫁接愈合过程中CiMYB46基因的克隆与表达分析[J]. 南京林业大学学报（自然科学版）, 2019, 43(5): 156-162.
[13]	祝友朋,刘宏莉,韩长志. 基于全基因组序列的核桃细菌性黑斑病菌分泌蛋白的预测及特征分析[J]. 南京林业大学学报（自然科学版）, 2019, 43(03): 17-22.
[14]	杨杰,孙璐,王思瑶,李影,翟睿,林香雨,詹亚光,尹静. 3个白桦细胞色素P450基因生物信息学及表达分析[J]. 南京林业大学学报（自然科学版）, 2018, 42(06): 27-34.
[15]	刘海琳,尹佟明. 全基因组测序技术研究及其在木本植物中的应用[J]. 南京林业大学学报（自然科学版）, 2018, 42(05): 172-178.

暴马桑黄MVD基因cDNA全长克隆及表达特性分析

The cloning and expression analysis of mevalonate pyrophosphate decarboxylase gene cDNA sequence from Sanghuangporus baumii

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 25

相关文章 15

编辑推荐

Metrics

本文评价

作者

读者

审稿