Cloning and Expression of GA20ox, NCED and IAA and Their Roles in Bulb Development of Fritillaria thunbergii
LI Zi-Ming1, FAN Xiao-Ping1, JIN Ze-Lan1, JIANG Jian-Ming2, WANG Zhi-An2, WANG Zhong-Hua1,*
1 Institute of Biotechnology, Zhejiang Wanli University, Ningbo 315100, China; 2 Zhejiang Institute of Traditional Chinese Medicine, Hangzhou 310023, China
Abstract：Gibberellin (GA) is a type of diterpene plant hormone, which plays an important role in the physiological development of plants. GA can promote the plant extension and affect the production of dry matter in sink organs. It can also activate α-amylase activity to promote catalyzes the degradation of energy storage substances and promotes the break of plant dormancy. For example, the increase of GA content can promote the germination of barley and make it enter the development stage earlier. GA also has an effect on fruit development and maturity, it can also delay leaf senescence and inhibit tuber formation. Gibberellin 20- oxidase (GA20ox) enzyme is the key rate-limiting enzyme in the final stage of GA synthesis pathway. It exercises catalytic regulation function in the final stage of GA synthesis and promotes the production of GA20 and GA9, those are precursors of active GA1 and GA4. GA20ox gene has a close influence on the activity of GA20ox enzyme and is closely related to GA. ABA (abscisic acid) is a 15-carbon sesquienene compound, which can effectively promote the development and growth of various plants. It plays an important role in seed germination, maturation and dormancy, fruit maturation. It can bring huge economic benefits and can effectively increase crop yields. There are 2 ways to synthesize ABA in plants, one is the indirect pathway, the C15 pathway, and the other is the direct pathway, which also can be called the carotenoid pathway. The synthesis of ABA in higher plant tissues is mainly achieved through indirect pathways. 9-cis epoxy carotenoid dioxygenase (NCED) is the rate-limiting enzyme in the process of indirect ABA synthesis and regulation. Auxin (IAA) is an endogenous hormone containing an unsaturated aromatic ring and an acetic acid side chain. IAA is a signal transduction compound that promotes and affects plant development and physiological changes. It plays an important role at every moment of development, affecting the division, differentiation and elongation of plant cells, as well as various physiological activities of plants, such as apical dominance, light orientation and gravity, and induces many division-related genes expression. Indoleacetic acid (AUX)/IAA auxin response protein can negatively regulate auxin synthesis. In this study, enzyme linked ELISA was used to determine the changes in GA, ABA, and IAA content in Fritillaria thunbergii during bulb development. As the bulb develops, GA content showed a downward trend. ABA showed an upward trend, IAA generally showed a downward trend. Using homologous cloning combined and RACE technology, Fritillaria thunbergii GA20ox (GenBank No. MW238816), NCED (GenBank No. MW238817) and IAA (GenBank No. MW238818) were successfully cloned. Bioinformatics analysis showed that the full length of the sequence of GA20ox gene was 1 490 bp, the open reading frame was 1 122 bp, and encoded 373 amino acids; the full length of the sequence of NCED gene was 2 270 bp , and the open reading frame was 1 800 bp, which encoded 599 amino acids; the full length of the sequence of IAA gene was 1 255 bp, with an open reading frame of 864 bp, encoding 287 amino acids. qRT-PCR analysis showed that the GA20ox, NCED, IAA genes expression in bulbs at different developmental stages correlated with GA, ABA, and IAA. The correlation analysis showed that GA20ox, NCED and GA, ABA content were significantly positively correlated (P<0.01), IAA and IAA content was a very significant negative correlation (P<0.01). This study provides a theoretical basis for the study of the functions of GA20ox, NCED, IAA genes in GA, ABA and IAA metabolism, and provides references for further exploring the molecular mechanisms of GA20ox, NCED and IAA genes in the bulb development process of F. thunbergii.
李梓铭, 范小平, 金泽兰, 江建铭, 王志安, 王忠华. 浙贝母GA20ox、NCED、IAA的克隆、表达及在鳞茎发育中的作用[J]. 农业生物技术学报, 2021, 29(9): 1710-1721.
LI Zi-Ming, FAN Xiao-Ping, JIN Ze-Lan, JIANG Jian-Ming, WANG Zhi-An, WANG Zhong-Hua. Cloning and Expression of GA20ox, NCED and IAA and Their Roles in Bulb Development of Fritillaria thunbergii. 农业生物技术学报, 2021, 29(9): 1710-1721.
[1] 白戈, 杨大海, 姚恒, 等. 2017. 烟草NtNCED基因的鉴定分析[J]. 分子植物育种, 15(10): 3907-3912. (Bai G, Yang D H, Yao H, et al.2017. Identification and analysis of tobacco NtNCED gene[J]. Molecular Plant Breeding, 15(10): 3907-3912.) [2] 陈敏敏, 顾俊杰, 沈强, 等. 2019. 郁金香鳞茎更新与植物激素变化关系[J]. 植物生理学报, 55(3): 301-309. (Chen M M, Gu J J, Shen Q, et al.2019. Relationship between tulip bulb renewal and plant hormone changes[J]. Acta Phytophysiology, 55(3): 301-309.) [3] 戴文珊, 王敏, 刘继红. 2020. 柠檬中超表达印度酸橘脱落酸合成基因CrNCED1增强脱水抗性[J]. 园艺学报, 47(3): 551-561. (Dai W S, Wang M, Liu J H.2020. Overexpression of citrus citrus abscisic acid synthetic gene CrNCED1 in lemons enhances dehydration resistance[J]. Acta Horticulturae Sinica, 47(3): 551-561.) [4] 丁苏芹, 晏姿, 李玺, 等. 2019. 香雪兰球茎发育的内源激素变化规律研究[J]. 中国农业科技导报, 21(9): 51-57. (Ding S Q, Yan Z, Li i, et al.2019. Study on the variation of endogenous hormones in the development of vanilla bulb[J]. Review of China Agricultural Science and Technology, 21(9): 51-57. [5] 方旭燕, 徐根娣, 刘鹏. 2005. 浙贝母组织培养技术的研究进展[J]. 安徽农业科学, 33(1): 124-125. (Fang X Y, Xu G D, Liu P.2005. Research progress of tissue culture technology of Fritillaria thunbergii[J]. Journal of Anhui Agricultural Sciences, 33(1): 124-125.) [6] 冯亚斌, 李沫宇, 吴秋丽, 等. 2017a. 浙贝母FPS基因的克隆及其在生物碱代谢中的调控作用研究[J]. 中草药, 48(5): 971-978. (Feng Y B, Li M Y, Wu Q L, et al.2017. The cloning of FPS gene of Fritillaria vulgaris and its regulation in alkaloid metabolism[J]. Chinese and Herbal Medicine, 48(5): 971-978.) [7] 冯亚斌, 施鑫磊, 俞信光, 等. 2017b. 浙贝母肌动蛋白基因的克隆及生物信息学分析[J]. 中成药, 39(1): 126-130. (Feng Y B, Shi X L, Yu X G, et al.2017. Cloning and bioinformatics analysis of the actin gene of Fritillaria from Zhejiang[J]. Chinese Patent Medicine, 39(1): 126-130.) [8] 高晓峰, 吕享, 吴彦秋, 等. 2016. 杜鹃兰假鳞茎发育中内源激素的含量变化[J]. 山地农业生物学报, 35(2): 34-39. (Gao X F, Lv X, Wu Y Q, et al.2016. Changes in endogenous hormones during the development of Rhododendron pseudobulbs[J]. Journal of Mountain Agriculture and Biology, 35(2): 34-39.) [9] 郭得平. 1996. 光敏素和激素对洋葱鳞茎形成的调控及其作用机制[J]. 植物生理学通讯, 32(3): 228-234. (Guo D P.1996. Regulation of onion bulb formation by photosensitizers and hormones and its mechanism[J]. Plant Physiology Communications, 32(3): 228-234.) [10] 黄炜, 师桂英, 黄彦玮, 等. 2018. 外源GA_3和ABA处理条件下‘兰州百合’鳞茎内源激素的变化[J]. 甘肃农业大学学报, 53(6): 129-136. (Huang W, Shi G Y, Huang Y W, et al.2018. Changes of endogenous hormones in the bulbs of 'Lanzhou Lily' under exogenous GA_3 and ABA treatments[J]. Journal of Gansu Agricultural University, 53(6): 129-136.) [11] 姜仲禹, 唐丽雪, 柳洪鹃, 等. 2020. 不同施钾量条件下甘薯块根形成的内源激素变化及其与块根数量的关系[J]. 作物学报, 46(11): 1750-1759. (Jiang Z Y, Tang L X, Liu H J, et al.2020. Changes of endogenous hormones in sweet potato tuber formation under different potassium application rates and its relationship with the number of tubers[J]. Acta Agronomica Sinica, 46(11): 1750-1759.) [12] 李丽. 2017. 两种贝母的组织培养及外源GA_3、ABA处理对甘肃贝母鳞茎休眠的促抑效应[D]. 硕士学位论文, 甘肃农业大学, 导师: 屈星, pp. 1-60. (Li L.2017. Tissue culture of two kinds of Fritillaria and exogenous GA3 and ABA treatments promote inhibitory effects on bulb dormancy of Fritillaria in Gansu[D]. Thesis for M.S., Gansu Agricultural University, Supervisor: Qu X, pp. 1-60.) [13] 刘秀莲, 王鲁彤, 戴德吉, 等. 2002. 激素组合对浙贝母小鳞茎切片培养的影响[J]. 浙江万里学院学报, 15(1): 67-68,55. (Liu X L, Wang L T, Dai D J, et al.2002. Effects of hormone combinations on the culture of small bulb slices of Fritillaria Zhejiang[J]. Journal of Zhejiang Wanli University, 15(1): 67-68, 55.) [14] 庞保亚, 李强, 任仲海. 2018. 黄瓜CsGA20ox1异源表达促进拟南芥植株发育[J]. 山东农业大学学报(自然科学版), 49(4): 578-584. (Pang B Y, Li Q, Ren Z H.2018. Cucumber CsGA20ox1 heterologous expression promotes Arabidopsis plant development[J]. Journal of Shandong Agricultural University (Natural Science Edition), 49(4): 578-584.) [15] 钱树林, 义鸣放. 2006. 不同生长发育时期唐菖蒲籽球内源激素变化的分析[J]. 河北农业大学学报, 29(2):9-12, 18. (Qian S L, Yi M F.2006. Analysis of endogenous hormones changes in gladiolus seed balls in different growth and development periods[J]. Journal of Hebei Agricultural University, 29(2):9-12, 18.) [16] 孙红梅, 李天来, 李云飞. 2004. 百合鳞茎发育过程中内源激素变化初探[J]. 华中农业大学学报, (z2): 277-281. (Sun H M, Li T L, Li Y F. 2004. Preliminary study on the changes of endogenous hormones during the development of lily bulb[J]. Journal of Huazhong Agricultural University, (z2): 277-281.) [17] 孙同玉, 祝娟, 孙鹏, 等. 2015. 西洋参赤霉素20-氧化酶基因的克隆与序列分析[J]. 中草药, 46(11): 1656-1660. (Sun T Y, Zhu J, Sun P, et al.2015. Cloning and sequence analysis of gibberellin 20-oxidase gene from American ginseng[J]. Chinese Herbal Medicine, 46(11): 1656-1660.) [18] 吴洁芳, 纪凯, 冷平, 等. 2010. 西葫芦果实CpNCED1基因3′端的克隆及其表达分析[J]. 中国农业大学学报, 15(5): 25-30. (Wu J F, Ji K, Leng P, et al.2010. Cloning and expression analysis of the 3′ end of CpNCED1 gene in summer squash fruit[J]. Journal of China Agricultural University, 15(5): 25-30.) [19] 夏宜平, 杨玉爱, 杨肖娥, 等. 2005. 郁金香更新鳞茎发育的碳同化物积累与内源激素变化研究[J]. 园艺学报, 32(2): 278-283. (Xia Y P, Yang Y A, Yang X E, et al.2005. Carbon assimilate accumulation and endogenous hormones changes in tulip renewal bulb development[J]. Acta Horticulturae Sinica, 32(2): 278-283.) [20] 肖月华, 叶应福, 冯怡, 等. 2006. 棉花赤霉素氧化酶同源基因GhGA20ox1在烟草中的功能表达(英文)[J]. 植物生理与分子生物学学报, 32(5): 563-569. (Xiao Y H, Ye Y F, Feng Y, et al.2006. Functional expression of cotton gibberellin oxidase homologous gene GhGA20ox1 in tobacco (English)[J]. Journal of Plant Physiology and Molecular Biology, 32(5): 563-569.) [21] 许俊旭, 李青竹, 李叶, 等. 2020. 石蒜鳞茎膨大过程中内源激素相关基因的差异表达研究[J]. 园艺学报, 47(6): 1126-1140. (Xu J X, Li Q Z, Li Y, et al.2020. Study on the differential expression of endogenous hormone-related genes during the bulb expansion of Lycoris radiata[J].Acta Horticulturae Sinica, 47(6): 1126-1140.) [22] 徐文娟, 李先恩, 孙鹏, 等. 2013. 滇重楼种子层积后脱落酸和赤霉素相关基因表达水平的研究[J]. 中草药, 44(3): 338-343. (Xu W J, Li X R, Sun P, et al.2013. Study on the expression levels of abscisic acid and gibberellin related genes after stratification of Rhizoma Paridis seeds[J]. Chinese Herbal Medicine, 44(3): 338-343.) [23] 张海燕, 段文学, 解备涛, 等. 2018. 不同时期干旱胁迫对甘薯内源激素的影响及其与块根产量的关系[J]. 作物学报, 44(1): 126-136. (Zhang H Y, Duan W X, Xie B T, et al.2018. Effects of drought stress in different periods on sweet potato endogenous hormones and its relationship with root tuber yield[J]. Acta Agronomica Sinica, 44(1): 126-136.) [24] 张旭红, 孙美玉, 李靖锐, 等. 2019. 东方百合'索邦'GA20ox的克隆及表达分析[J]. 园艺学报, 46(1): 74-86. (Zhang X H, Sun M Y, Li J R, et al.2019. Cloning and expression analysis of Oriental Lily 'Sorbon' GA20ox[J]. Acta Horticulturae Sinica,46(1): 74-86.) [25] 张玉喜, 张文超, 李玉娥, 等. 2014. 赤霉素氧化酶PsGA20ox基因参与低温诱导的牡丹内休眠解除[J]. 华北农学报, 29(3): 22-26. (Zhang Y X, Zhang W C, Li Y E, et al.2014. Gibberellin oxidase PsGA20ox gene participates in low temperature-induced release of dormancy in tree peony[J]. North China Agricultural Journal, 29(3): 22-26.) [26] Carrera E.1999. Feedback control and diumal regulation of giberellin 20-xidase transript levels in potato[J]. Plant Physiology, 119(2): 765-773. [27] Endo A, Takahashi H, Sawada Y, et al.2008. Drought induction of Arabidopsis 9-cis-epoxycarotenoid dioxygenase occurs in vascular parenchyma cells[J]. Plant physiology, 147(4): 1984-1993. [28] Eriksson M E, Israelsson M, Olsson O, et al.2000. Increased gibberellin biosynthesis in transgenic trees promotes growth, biomass production and xylem fiber length[J]. Nature Biotechnology: The Science and Business of Biotechnology, 18(7): 784-788. [29] He R R, Zhuang Y, Cai Y M, et al.2018. Overexpression of 9-cis-epoxycarotenoid dioxygenase cisgene in grapevine increases drought tolerance and results in pleiotropic effects[J]. Frontiers in Plant Science, 9(1): 970-985. [30] Huang S.1998. Overexpression of 20-oxidase confers a gibberellin-overproduction phenotype in Arabidopsis[J]. Plant Physiology, 118(3): 773-781. [31] Nicholas C M.1976. Polarity and localization of auxin movement in the hepatic, marchantia polymorpha[J]. American Journal of Botany, 63(5): 526-531. [32] Rodrigo M-J, Alquezar B, Zacarías L.2006. Cloning and characterization of two 9-cis-epoxycarotenoid dioxygenase genes, differentially regulated during fruit maturation and under stress conditions, from orange (Citrus sinensis L. Osbeck)[J]. Journal of Experimental Botany, 57(3): 633-643. [33] Sankar B, Jaleel C A, Manivannan P, et al.2007. Alterations in carbohydrate metabolism and enhancement in tuber production in white yam (Dioscorea rotundata Poir.) under triadimefon and hexaconazole applications[J]. Plant Growth Regulation, 53(1): 7-16. [34] Sasaki A, Ashikari M, Ueguchi-Tanaka M, et al.2002. A mutant gibberellin-synthesis gene in rice[J]. Nature, 416(6882): 701-702. [35] Su L, Bassa C, Audran C, et al.2014. The auxin Sl-IAA17 transcriptional repressor controls fruit size via the regulation of endoreduplication-related cell expansion[J]. Plant & Cell Physiology, 55(11): 1969-1976. [36] Sun L, Yuan B, Zhang M, et al.2012. Fruit-specific RNAi mediated suppression of SlNCED1 increases both lycopene and E-carotene contents in tomato fruit[J]. Journal of Experimental Botany, 63(8): 3097-3108. [37] Teitz A, Ludwing M, Dingkuhn M.1981. Effect of absicsic acid on transport of assimilates in barley[J]. Planta, 152(6): 557-561. [38] Tiwari S B, Wang X J, Hagen G, et al.2001. AUX/IAA proteins are active repressors, and their stability and activity are modulated by auxin[J].The Plant cell, 13(12): 2809-2822. [39] Ulmasov T, Murfett J, Hagen G, et al.1997. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements[J]. The Plant Cell, 9(11): 1963-1971. [40] Xiao Y H, Ye Y F, Feng Y, et al.2006. Functional expression of the cotton gibberellic acid oxidase homolgous gene GhGA20ox1 in tobacco[J]. Journal of Plant Physiology and Molecular Biology. 32(5):563-569.
