Effect of Expression of Aeβ-AS gene from Aralia elata on the Content of Saponins in Nicotiana tabacum L.

doi:10.7525/j.issn.1673-5102.2023.05.014

Abstract

Abstract:

To investigate the effect of β-amyrin synthase gene（Aeβ-AS） on triterpenoid saponin synthesis of Aralia elata， the Aeβ-AS gene was cloned and genetically transformed into tobacco. The expression difference of Aeβ-AS gene in different parts of transgenic tobacco was analyzed， and the expression levels of Aeβ-AS gene， upstream and downstream key enzyme genes and total triterpene content were detected respectively. The results showed that the plant overexpression vector PROKⅡ-Aeβ-AS was successfully constructed and transferred into wild type tobacco， and seven transgenic lines were obtained and expressed at the mRNA level， and the expression level in leaves was higher than that in roots and stems. In transgenic tobacco， the expression levels of Aeβ-AS gene and its upstream and downstream key enzyme genes were higher than those of wild type， and the relative expression levels of NtFPS and NtSS genes of strain L21 were the highest， and the relative expression levels of NtSE and Aeβ-AS genes of strain L30 were the highest. Compared with wild-type tobacco， the total triterpene content of GM tobacco was significantly increased（1.1-1.6 times）. The results showed that synthesis of Aeβ-AS gene and heterologous transformation in tobacco could significantly increase the total triterpene content in transgenic tobacco.

Key words: Aralia elata, Aeβ-AS gene, Nicotiana tabacum, total triterpenoids content

CLC Number:

Q949.763.2

Qingqing HUO, Yuxin XIA, Jiale LI, Wei HAN, Shuya ZHANG, Zhe ZHANG, Meiling XIA, Wenhua GUO, Xiangling YOU. Effect of Expression of Aeβ-AS gene from Aralia elata on the Content of Saponins in Nicotiana tabacum L.[J]. Bulletin of Botanical Research, 2023, 43(5): 779-786.

Figures/Tables 8

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References 27

1	孙涛，张国荣，王彦，等.龙牙楤木的化学成分、药理作用和食用价值研究进展［J］.人参研究，2009，21（1）： 24-25.
	SUN T， ZHANG G R， WANG Y，et al.Research progress on chemical constituents，pharmacological effects and edible value of Aralia elata ［J］.Ginseng Research，2009，21（1）：24-25.
2	王丽岩，肖洪彬，王鸣慧.辽东楤木叶总皂苷抗急性肝损伤的实验研究［J］.中医药信息，2009，26（2）：29-30.
	WANG L Y， XIAO H B， WANG M H.Effect of total saponins of Aralia elata on acute liver injury［J］.Information on Traditional Chinese Medicine，2009，26（2）：29-30.
3	韩玉英.龙牙楤木叶总皂甙的遗传毒性及抗肿瘤作用研究［D］.哈尔滨：黑龙江中医药大学，2002.
	HAN Y Y.Study on genotoxicity and antitumor effect of total saponins from leaves of Aralia elata ［D］.Harbin：Heilongjiang University of Traditional Chinese Medicine，2002.
4	LIU Y， LIU J F， LIU Z H，et al.The antitumor effects of araloside a extracted from the root bark of Aralia elata on human kidney cancer cell lines［J］.African Journal of Pharmacy and Pharmacology，2011，5（4）：462-467.
5	任美萍，刘艳，李蓉，等.楤木皂苷抗炎、镇痛作用的实验研究［J］.泸州医学院学报，2012，35（2）：133-135.
	REN M P， LIU Y， LI R，et al.Experimental study on anti-inflammatory and analgesic effects of Aralia elata saponins［J］.Journal of Luzhou Medical College，2012，35（2）：133-135.
6	宋少江，徐绥绪，曹颖林.辽东楤木总皂苷降血糖作用研究［J］.中药研究与信息，2005，7（5）：7-9.
	SONG S J， XU S X， CAO Y L.Study on hypoglycemic effect of total saponins of Aralia elata ［J］.Research & Information on Traditional Chinese Medicine，2005，7（5）：7-9.
7	YOSHIKAWA M， MATSUDA H， HARADA E，et al.Elatoside E，a new hypoglycemic principle from the root cortex of Aralia elata Seem.：structure-related hypoglycemic activity of oleanolic acid glycosides［J］.Chemical and Pharmaceutical Bulletin，1994，42（6）：1354-1356.
8	郑玲玲，裴凌鹏.楤木属植物研究进展［J］.中国民族医药杂志，2010，16（6）：57-59.
	ZHENG L L， PEI L P.Research progress of Aralia［J］.Journal of Medicine & Pharmacy of Chinese Minorities，2010，16（6）：57-59.
9	THIMMAPPA R， GEISLER K， LOUVEAU T，et al.Triterpene biosynthesis in plants［J］.Annual Review of Plant Biology，2014，65（1）：225-257.
10	AUGUSTIN J M， KUZINA V， ANDERSEN S B，et al.Molecular activities，biosynthesis and evolution of triterpenoid saponins［J］.Phytochemistry，2011，72（6）：435-457.
11	ZHAO Y J， LI C.Biosynthesis of plant triterpenoid saponins in microbial cell factories［J］.Journal of Agricultural and Food Chemistry，2018，66（46）：12155-12165.
12	MIETTINEN K， IÑIGO S， KREFT L，et al.The TriForC database：a comprehensive up-to-date resource of plant triterpene biosynthesis［J］.Nucleic Acids Research，2018，46（D1）：D586-D594.
13	SUZUKI H， ACHNINE L， XU R，et al.A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula ［J］.The Plant Journal，2002，32（6）：1033-1048.
14	KUSHIRO T， SHIBUYA M， EBIZUKA Y.β-amyrin synthase—cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants［J］.European Journal of Biochemistry，1998，256（1）：238-244.
15	SHIBUYA M， KATSUBE Y， OTSUKA M，et al.Identification of a product specific β-amyrin synthase from Arabidopsis thaliana ［J］.Plant Physiology and Biochemistry，2009，47（1）：26-30.
16	HAYASHI H， HUANG P Y， KIRAKOSYAN A，et al.Cloning and characterization of a cDNA encoding β- amyrin synthase involved in glycyrrhizin and soyasaponin biosyntheses in licorice［J］.Biological and Pharmaceutical Bulletin，2001，24（8）：912-916.
17	袁丁，焦梦婷，许成，等.竹节参β-香树素合成酶基因克隆及生物信息学分析［J］.中草药，2020，51（19）：5028-5034.
	YUAN D， JIAO M T， XU C，et al.Cloning and bioinformatics analysis of β-balsamine synthetase gene of Panax japonicus ［J］.Chinese Traditional and Herbal Drugs，2020，51（19）：5028-5034.
18	WU Y， ZOU H D， CHENG H，et al.Cloning and characterization of a β-amyrin synthase gene from the medicinal tree Aralia elata （Araliaceae）［J］.Genetics and Molecular Research，2012，11（3）：2301-2314.
19	赵微，尹静，詹亚光，等.温度胁迫对白桦悬浮细胞中三萜积累及防御酶活性的影响［J］.中国生物工程杂志，2013，33（2）：34-40.
	ZHAO W， YIN J， ZHAN Y G，et al.Effects of temperature stress on triterpenoid accumulation and defense enzyme activities in Betula alba suspension cells［J］.China Biotechnology，2013，33（2）：34-40.
20	王紫晴，李飞洁，郭玉婷，等.硫化氢对白桦悬浮细胞中多胺和次生代谢物积累的影响［J］.南京林业大学学报（自然科学版），2017，41（6）：26-32.
	WANG Z Q， LI F J， GUO Y T，et al.Effects of hydrogen sulfide on accumulation of polyamines and secondary metabolites in Betula alba suspension cells［J］.Journal of Nanjing Forestry University （Natural Sciences Edition），2017，41（6）：26-32.
21	张玉文，伊恒杰，赵帅，等.藤茶香树脂醇合成酶基因编码区克隆及表达分析［J］.西北植物学报，2017，37（3）：428-435.
	ZHANG Y W， YI H J， ZHAO S，et al.Cloning and expression analysis of Amyrin synthase gene coding sequence in Ampelopsis grossedentata ［J］.Acta Botanica Boreali-Occidentalia Sinica，2017，37（3）：428-435.
22	JO H J， HAN J Y， HWANG H S，et al. β-Amyrin synthase （EsBAS） and β-amyrin 28-oxidase （CYP716A244） in oleanane-type triterpene saponin biosynthesis in Eleutherococcus senticosus ［J］.Phytochemistry，2016，135：53-63.
23	UM Y， JIN M L， LEE D Y，et al.Functional characterization of the β-amyrin synthase gene involved in platycoside biosynthesis in Platycodon grandiflorum［J］.Horticulture，Environment，and Biotechnology，2017，58（6）：613-619.
24	沈湛云，刘春生，黄建梅，等.甘草β-香树酯醇合成酶的多态性对其催化效率的研究［J］.中国中药杂志，2010，35（22）：2941-2944.
	SHEN Z Y， LIU C S， HUANG J M，et al.Study on the effect of β-vanillin synthetase polymorphism on the catalytic efficiency of Glycyrrhiza glabra ［J］.China Journal of Chinese Materia Medica，2010，35（22）：2941-2944.
25	孙化鹏，钟晓红，徐子健，等.洋常春藤β-AS基因的全长克隆与表达分析［J］.分子植物育种，2018，16（3）：733-739.
	SUN H P， ZHONG X H， XU Z J，et al.Cloning and expression analysis of β-AS gene in Hedera helix L.［J］.Molecular Plant Breeding，2018，16（3）：733-739.
26	ZHAO C， XU T H， LIANG Y L，et al.Functional analysis of β-amyrin synthase gene in ginsenoside biosynthesis by RNA interference［J］.Plant Cell Reports，2015，34（8）：1307-1315.
27	陈勤，刘美佳，刘迪秋，等. PjFPS和Pjβ-AS双基因协同作用对珠子参皂苷生物合成的影响［J］.浙江农业学报，2022，34（3）：428-436.
	CHEN Q， LIU M J， LIU D Q，et al.Effects of synergy by double genes of PjFPS and Pjβ-AS on biosynthesis of Panax japonicus saponins［J］.Acta Agriculturae Zhejiangensis，2022，34（3）：428-436.

引物名称 Primer names	引物序列5′→3′ Primer sequences 5′→3′
Aeβ-AS-F	gagaaacacgggggactctagaATGTGGAAGCTTAAGATAGCGAAA
Aeβ-AS-R	gggaaattcgagctcggtaccTTAGGTGCCTAGGGACGGTAAT
pROKⅡ-F	AAGACCGGCAACAGGATTC
pROKⅡ-R	CGCACAATCCCACTATCCTT
RT-NtActin-F	CCTGAGGTCCTTTTCCAACCA
RT-NtActin-R	GGATTCCGGCAGCTTCCATT
RT-Aeβ-AS-F	CTTCCTATGCACCCAGCTAAA
RT-Aeβ-AS-R	CCCAGAGCAGGTCTTGTATTT
RT-NtFPS-R	TACTTCTCCGCAACCACATC
RT-NtFPS-R	GGACAATCCGGCGATGAATA
RT-NtSS-R	GTACCCAAGTGCCGTATGTT
RT-NtSS-R	TTGCCATGACCTGTGGAATAG
RT-NtSE-R	CAGATCCTTCGCCCATCTTATTT
RT-NtSE-R	CAGCTGGCATGCTCCTATTT

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