Abstract：Suspension culture of Eriobotrya japonica (loquat) can produce rich pentacyclic triterpenoids. Amyrine synthase (AS), a key enzyme in the triterpenoid synthesis pathway, determines the branching point of major metabolism and secondary metabolism. But the function of the AS in Eriobotrya japonica has not been reported before. In this study, using the AS overexpression plasmid pDONR207-AS as the template, the positive open reading frame of the AS gene was amplified by PCR, then ligated into the fusion expression vector pBWA(V)HS-GFP after digestion to construct the expression vector pBWA(V)HS-AS-GFP-Tnos. Next, using loquat suspension cells (homologous system with AS isolated) as receptor materials, transient expression was carried out under the mediation of Agrobacterium tumefaciens GV3101. Finally, the subcellular localization of AS protein were analyzed by imaging of co-transformed GFP under laser confocal microscopy, and the catalytic products of AS protein were analyzed by conjoint analysis of gas chromatography-mass spectrometry. Results were as follows: The subcellular localization of AS protein was in the endoplasmic reticulum in loquat. And the catalytic products of AS protein contained α-amyrin and β-amyrin, which indicated that AS of loquat belonged to the multifunctional enzyme. This study provides a reference for improvement of the synthesis of pentacyclic triterpenoid and for the further transgenic application in suspension cells of loquat.
李惠华, 曾碧玉, 王伟, 常强, 苏明华. 枇杷悬浮细胞AS基因的瞬时表达载体构建与亚细胞定位[J]. 农业生物技术学报, 2019, 27(11): 1923-1932.
LI Hui-Hua, ZENG Bi-Yu, WANG Wei, CHANG Qiang, SU Ming-Hua. Transient Expression Vector Construction of AS Gene from Suspension Cells of Eriobotrya japonica and Its Subcellular Localization. 农业生物技术学报, 2019, 27(11): 1923-1932.
[1] 陈大华, 叶和春, 李国凤,等. 2000. 植物类异戊二烯代谢途径的分子生物学研究进展[J].植物学报, 42(6): 551-558. (Chen D H, Ye H C, Li G F, et al.2000. Advances in molecular biology of plant isoprenoid metabolic pathway[J]. Acta Botanica Sinica, 42(6): 551-558.) [2] 陈建, 赵德刚. 2004. 植物萜类生物合成相关酶类及其编码基因的研究进展[J]. 分子植物育种, 2(6): 757-764. (Chen J, Zhao D G.2004. Research advances on the enzymes and their coding gene involved in plant terpene biosynthesis[J]. Molecular Plant Breeding, 2(6): 757-764.) [3] 董燕妮, 邓琼仙, 王永清. 2008. 我国枇杷种质资源与育种的研究进展[J]. 亚热带农业研究, 4(2): 91-96. (Dong Y N, Deng Q X, Wang Y Q.2008. Advances in germplasm resources and breeding of loquat in China[J]. Subtropical Agriculture Research, 4(2): 91-96.) [4] 李惠华, 刘小英, 王伟, 等. 2013. 适合于悬浮培养的枇杷愈伤组织诱导及状态调控[J]. 亚热带植物科学, 42(4): 309-313. (Li H H, Liu X Y, Wang W, et al.2013. Callus inductin and state regulation in Eriobotrya japonica for suspension culture[J]. Subtropical Plant Science, 42(4): 309-313.) [5] 李惠华, 常强, 王伟, 等. 2018. 基于转录组测序的枇杷五环三萜合成途径差异基因的分析[J]. 农业生物技术学报, 26(2): 222-233. (Li H H, Chang Q, Wang W, et al.2018. Differential genes analysis of triterpenoid synthesis pathway based on loquat transcriptome sequencing[J]. Journal of Agricultural Biotechnology, 26(2): 222-233.) [6] 李惠华, 刘小英, 常强, 等. 2015. 枇杷细胞悬浮培养生产熊果酸的调控[J]. 热带作物学报, 36(7): 1247-1253. (Li H H, Liu X Y, Chang Q, et al.2015. Regulation of ursolic acid content in suspension culture of Eriobotrya japonica L[J]. Chinese Journal of Tropical Crops, 36(7): 1247-1253.) [7] 李名扬, 蒋建国, 罗静. 1991. 根癌农杆菌T-DNA在枇杷子叶外植体中的转移与表达[J]. 西南农业大学学报, 13(4): 442-445. (Li M Y, Jiang J G, Luo J.1991. Transfer and expression of Agrobacterium tumefaciens harboured T-DNA in cultrured co-tyledon explants of Eriobotrya japonica Lindl[J]. Journal of Southwest Agricultural University, 13(4): 442-445.) [8] 刘春朝, 王玉春, 赵兵, 等. 1999.生物诱导子调节植物组织次生代谢的研究[J]. 植物学通报, 16(2): 131-137. (Liu C Z, Wang Y C, Zhao B, et al.1999. Regulation of plant tissue secondary metabolism by biotic elicitors[J]. Chinese Bulletin of Botany, 16(2): 131-137.) [9] 刘维, 刘浩, 董双玉, 等. 2017. 水稻叶鞘原生质体转化体系的构建及Pik-H4和AvrPik-H4蛋白的瞬时表达[J]. 中国农业科学, 50(23): 4575-4584. (Liu W, Liu H, Dong S Y, et al.2017. Construction of rice leaf sheath protoplast transformation system and transient expression of Pik-H4 and AvrPik-H4 proteins[J]. Scientia Agricultura Sinica, 50(23): 4575-4584.) [10] 王和勇, 罗恒, 孙敏. 2004. 诱导子在药用植物细胞培养中的应用[J]. 中草药, 35(8): 附3-7.(Wang H Y, Luo H, Sun M. 2004. Application of elicitor to cell culture of medicinal plants[J]. Chinese Traditional and Herbal Drugs, 35(8): Appendix 3-7. [11] 王华忠, 陈雅平, 陈佩度. 2007. 植物瞬间表达系统与功能基因组学研究[J]. 生物工程学报, 23(3): 367-374. (Wang H Z, Chen Y P, Chen P D.2007. Plant transient expression system in functional genomics[J]. Chinese Journal of Biotechnology, 23(3): 367-374.) [12] 吴颖. 2011.辽东楤木三萜皂苷合成相关基因克隆及功能研究[D]. 博士学位论文, 吉林大学, 导师: 原亚萍, pp. 42-100. (Wu Y.2011. Cloning and functional analysis of triterpenoid saponin related genes in Aralia elats[D]. Thesis for Ph.D., Jilin University, Supervisor: Yuan Y P, pp. 42-100.) [13] 肖成江, 程汉, 祝建顺, 等. 2011. 橡胶树细胞悬浮系电激转化体系的初步研究[J].热带作物学报, 32(2): 245-250. (Xiao C J, Cheng H, Zhu J S, et al.2011.Transformation of Hevea suspension cells by electroporation[J]. Chinese Journal of Tropical Crops, 32(2): 245-250.) [14] 晏琼, 胡宗定, 吴建勇. 2006. 生物和非生物诱导子对丹参毛状根培养生产丹参酮的影响[J]. 中草药, 37(2): 262-265. (Yang Q, Hu Z D, Wu J Y.2006. Influence of biotic and abiotic elicitors on production of tanshinones in Salvia miltiorrhiza hairy root culture[J]. Chinese Traditional and Herbal Drugs, 37(2): 262-265. [15] 杨志武, 王永清, 刘娟, 等. 2011. 根癌农杆菌介导的枇杷花药胚遗传转化体系研究[C]//第五届全国枇杷学术研讨会论文集, 中国园艺学会枇杷分会出版, 广州, pp. 63-67. (Yang Z W, Wang Y Q, Liu J, et al.2011. Studies on Agrobacterium tumefacies-mediated genetic transformation system of anther-derived embryos of Eriobotrya japonica Lindl[C]//Proceedings of 5th symposium in Loquat section, Chinese society for horticultural science. Loquat section of Chinese society for horticultural science, Guangzhou, pp. 63-67.) [16] 赵云生, 万德光, 陈新, 等. 2009. 五环三萜皂苷生物合成与调控的研究进展[J]. 中草药, 40(2): 327-330. (Zhao Y S, Wan D G, Chen X, et al.2009. Advances in studies on biosynthesis and regulation of pentacyclic triterpenoid saponin[J]. Chinese Traditional and Herbal Drugs, 40(2): 327-330.) [17] Akhond M A Y, Machray G C.2009. Development of an efficient transient gene expression assay based on tobacco (Nicotiana tabacum var. Xanthi) male gametophytes[J]. Plant Tissue Culture and Biotechnology, 19(1): 9-23. [18] Banno N, Akihisa T, Tokuda H, et al.2004. Triterpene acids from the leaves of perilla frutescens and their anti-inflammatory and antitumor-promoting effects[J]. Bioscience Biotechnology and Biochemistry, 68(1): 85-90. [19] Brendolise C,Yauk Y K, Eberhard E D, et al.2011. An unusual plant triterpene synthase with predominant α-amyrin-producing activity identified by characterizing oxidosqualene cyclases from Malus×domestica[J]. The FEBS Journal, 2011, 278(14): 2485-2499. [20] Chang C T, Huang S S, Lin S S.2011. Anti-inflammatory activities of tormentic acid from suspension cells of Eriobotrya Japonica ex vivo and in vivo[J]. Food Chemistry, 127(3): 1131-1137.) [21] Fujikawa Y, Nampo T, Mori M, et al.2018. Fluorescein diacetate (FDA) and its analogue as substrates for Pi-class glutathione S-transferase (GSTP1) and their biological application[J]. Talanta, 179(3): 845-852. [22] Hayashi H, Huang P Y, Inoue K.2003. Up-regulation of soyasaponin biosynthesis by methyl jasmonate in culthure cells of Glycyrrhiza glabra[J]. Plant and Cell Physiology, 44(4): 404-411. [23] Hayashi H, Huang P Y, Kirakosyan A, et al.2001. Cloning and characterization of a cDNA encoding beta-amyrin synthase involved in glycyrrhizinand soyasaponin biosyntheses in licorice[J]. Biological & Pharmaceutical Bulletin, 24(8): 912-916. [24] Hayashi H, Huang P Y, Takada S, et al.2004. Differential expression of three oxidosqualene cyclase mRNAs in Glycyrrhizaglabra[J]. Biological & Pharmaceutical Bulletin, 27(7): 1086-1092. [25] Ho H Y, Liang K Y, Lin W C, et al.2010. Regulation and improvement of triterpene formation in plant cultured cells of Eriobotrya japonica Lindl[J]. Journal of Bioscience and Bioengineering, 110(5): 588-592.) [26] Husselstein-Muller T, Schaller H, Benveniste P.2001. Molecular cloning and expression in yeast of 2,3-oxidosqualene-triterpenoid cyclases from Arabidopsis thaliana[J]. Plant Molecular Biology, 45(1): 75-92. [27] Jang S M, Yee S T, Choi J, et al.2009. Ursolic acid enhances the cellular immune system and pancreatic beta-cell function in streptozotocin-induced diabetic mice fed a high-fat diet[J]. International Immunopharmacology, 9(1): 113-119. [28] Kushiro T, Shibuya M, Masuda K, et al.2000. A novel multifunctional triterpene synthase from Arabidopsis thaliana[J]. Tetrahedron Letters, 41(40): 7705-7710. [29] Levy M, Rachmilevitch S, Abel S.2005. Transient Agrobacterium-mediated gene expression in the Arabidopsis hydroponics root system for subcellular localization studies[J]. Plant Molecular Biology Reporter, 23(2): 179-184. [30] Li H H, Su M H, Yao D H, et al.2017. Anti-hepatocellular carcinoma activity of tormentic acid derived from suspension cells of Eriobotrya japonica (Thunb.) Lindl[J]. Plant Cell, Tissue and Organ Culture, 130(2): 427-433. [31] Lu Q Y, Zhang X, Yang J, et al.2017. Triterpenoid-rich loquat leaf extract induces growth inhibition and apoptosis of pancreatic cancer cells through altering key flux ratios of glucose metabolism[J]. Metabolomics, 13(4): 39. [32] Machado D G, Bettio L E, Cunha M P, et al.2009. Antidepressant-like effect of the extract of Rosmarinus officinalis in mice: Involvement of the monoaminergic system[J]. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 33(4): 642-650. [33] Mravec J, Skupa P, Bailly A L, et al.2009. Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter[J]. Nature, 459(25): 1136-1142. [34] Olivier V, Susana R, Pere M, et al.2003. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus[J]. The Plant Journal, 33(5): 949-956. [35] Saimaru H, Orihara Y, Tansakul P, et al.2007. Production of triterpene acidsby cell suspension cultures of Olea europaea[J]. Chemical & Pharmaceutical Bulletin, 55(5): 784-788. [36] Schillberg S, Zimmermann S, Priifer D, et al.1998. Transient gene expression in plant protoplasts[M]. //In: Cunningham C, Porter A J R. (eds) Recombinant Proteins from Plants. Methods in Biotechnology, vol 3. Humana Press, 1998: 165-175. [37] Schledzewski K, Mendel R R.1994. Quantitative transient gene expression: Comparison of the promoters for maize polyubiquitin1, rice actin1, maize-derived Emu and CaMV 35S in cells of barley, maize and tobacco[J]. Transgenic Research, 3(4): 249-255. [38] Shih C C, Ciou J L, Lin C H, et al.2013. Cell suspension culture of Eriobotrya japonica regulates the diabetic and hyperlipidemic sign of High-Fat-Fed mice[J]. Molecules, 18(3): 2726-2753. [39] Töpfer R, Prols M, Schell J, et al.1988. Transient gene expression in tobacco protoplasts: Ⅱ. Comparison of the reporter gene systems for CAT, NPTⅡ, and GUS[J]. Plant Cell Reports, 7(4): 225-228. [40] Yoo S D, Cho Y H, Sheen J.2007. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis[J]. Nature Protocols, 2(7): 1565-1572. [41] Zhang C H, Yan Q, Cheuk W K, et al.2004. Enhancement of tanshinone production in Salvia miltiorrhiza hairy root culture by Ag+ elicitation and nutrient feeding[J]. Planta Medica, 70(2): 147-151.
