桂花OfNCED3调控转基因烟草叶片类胡萝卜素和叶绿素的合成

doi:10.16420/j.issn.0513-353x.2022-0330

摘要/Abstract

摘要：

从桂花（Osmanthus fragrans）‘堰虹桂’转录组数据库中筛选获得OfNCED3基因序列。氨基酸序列分析发现OfNCED3具有NCED家族特有的2个保守结构域MIAHPKxDP和HDFAITE以及4个保守的组氨酸残基。进化树分析发现OfNCED3与番茄SlNCED1聚为一支。实时荧光定量PCR结果表明，桂花开放过程中，OfNCED3在类胡萝卜素含量较高的‘堰虹桂’花瓣中的表达量显著低于类胡萝卜素含量较低的‘玉玲珑’，推测OfNCED3可能与‘堰虹桂’和‘玉玲珑’花瓣中类胡萝卜素含量差异有关。通过农杆菌介导的遗传转化方法获得过表达OfNCED3烟草株系。利用分光光度计和超高效液相（ultra high performance liquid chromatography，UPLC）测定叶绿素、类胡萝卜素和ABA的含量，并采用实时荧光定量PCR分析类胡萝卜素通路上相关基因的表达差异，结果发现异源过表达OfNCED3导致转基因烟草叶片中叶绿素和类胡萝卜素含量下降，ABA含量增加，类胡萝卜素合成基因的表达量明显上调，且下游合成基因上调幅度高于上游合成基因。本研究表明OfNCED3能降解类胡萝卜素，促进内源ABA合成。

关键词: 桂花, NCED, 类胡萝卜素, 叶绿素, ABA

Abstract:

In this study，the OfNCED3 cDNA sequence were acquired based on the transcriptional data of Osmanthus fragrans‘Yanhong Gui’. The multiple sequence alignment analysis showed that OfNCED3 had two conservative domain（MIAHPKxDP and HDFAITE）characteristics of the NCED family and four conserved histidine residues. Phylogenetic analysis indicated that OfNCED3 is grouped together with SlNCED1. The results of real-time quantitative PCR showed that the expression of OfNCED3 in the petals of‘Yanhong Gui’with higher carotenoid content was significantly lower than that of‘Yu Linglong’with lower carotenoid content during flower opening process. It is speculated that OfNCED3 may be related to the difference of carotenoid contents between‘Yanhong Gui’and‘Yu Linglong’petals. Transgenic tobacco lines overexpressing OfNCED3 were obtained by an Agrobacterium-mediated method. The contents of chlorophyll，carotenoid and ABA were determined by using spectrophotometer and ultra high performance liquid chromatography（UPLC），and the expression levels of genes related to carotenoid pathway were analyzed via real-time quantitative PCR. The overexpression of OfNCED3 gene expression resulted in a significant decrease of chlorophyll and carotenoid contents and a increase of endogenous ABA content. The expression of carotenoid synthesis genes was significantly up-regulated，and the downstream synthesis genes were more up-regulated than the upstream synthesis genes. This research suggests OfNCED3 can promote the synthesis of ABA by degrading carotenoids in plants.

Key words: Osmanthus fragrans, NCED, carotenoids, chlorophyll, ABA

中图分类号:

S685.13

杜艳霞, 王艺光, 肖政, 董彬, 方遒, 钟诗蔚, 杨丽媛, 赵宏波. 桂花OfNCED3调控转基因烟草叶片类胡萝卜素和叶绿素的合成[J]. 园艺学报, 2023, 50(6): 1284-1294.

DU Yanxia, WANG Yiguang, XIAO Zheng, DONG Bin, FANG Qiu, ZHONG Shiwei, YANG Liyuan, ZHAO Hongbo. Ectopic Overexpression of OfNCED3 Regulates the Synthesis of Carotenoids and Chlorophylls in Transgenic Tobacco Leaves[J]. Acta Horticulturae Sinica, 2023, 50(6): 1284-1294.

图/表 8

表1 实时荧光定量PCR特异性引物

Table 1 Specific primers for real-time quantitative PCR

基因 Gene	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
OfNCED3	TTCAAAGCCGCAATGGAATTTTATT	GATAGATTCTGGGATTTTCCCTGTC
OfACT	CCCAAGGCAAACAGAGAAAAAAT	ACCCCATCACCAGAATCAAGAA
Nt26S-RNA	GAAGAAGGTCCCAAGGGTTC	TCTCCCTTTAACACCAACGG

图1 OfNCED3氨基酸序列与其他物种NCED的多重序列比对 Of：桂花；Sl：番茄；At：拟南芥；Nt：烟草；Cs：甜橙。方框表示NCED家族特有保守结构域序列MIAHPKxDP和HDFAITE；▼表示4个活性保守的组氨酸位点。

Fig. 1 Multiple alignment of amino acid sequences of OfNCED3 and NCEDs in other plants Of：Osmanthus fragrans；Sl：Solanum lycopersicum；At：Arabidopsis thaliana；Nt：Nicotiana tabacum；Cs：Citrus sinensis. The NCED motifs of MEAHPKXDP and HDFAITK are boxed. Four conserved histidines required for activity are marked by ▼.

图2 桂花OfNCED3蛋白与其他物种NCED蛋白系统进化树分析

Fig. 2 Phylogenetic tree of NCED proteins in Osmanthus fragrance and other plants

图3 OfNCED3基因在‘堰虹桂’和‘玉玲珑’桂花花开放进程中的表达量 S1：圆珠期；S2：顶壳期；S3：铃梗期；S4：初开期。 ** 代表不同品种之间差异显著（P < 0.01）；*** 代表不同品种之间差异极显著（P < 0.001）。

Fig. 3 Expression level of OfNCED3 gene in flower opening process of Osmanthus fragrans‘Yanhong Gui’and‘Yu Linglong’ S1：Bead stage；S2：Apical shell stage；S3：Boll stem stage；S4：Initial flowering stage. ** indicate statistically significant difference (P < 0.01); *** indicate extremely significant difference (P < 0.001).

图4 OfNCED3过表达载体（A）、转化烟草表型（B）及其表达水平（C）

Fig. 4 Overexpression vector (A), transformed tobacco phenotype (B) and expression level (C) of OfNCED3 in transgenic tobacco plants

图5 过表达OfNCED3对烟草叶片中叶绿素（A）、类胡萝卜素（B）和ABA（C）影响不同小写字母表示差异显著（P < 0.05）。下同。

Fig. 5 Effects of overexpression of OfNCED3 gene on chlorophyll（A）, carotenoid（B）and ABA（C）content in tobacco leaves Different lowercase letters indicate significant differences（P < 0.05）. The same below.

图6 转OfNCED3烟草植株中类胡萝卜素通路上相关基因的表达量

Fig. 6 Relative expression levels of carotenoid biosynthetic genes in tobacco leaves of the control and transgenic lines

表2 转OfNCED3烟草植株中类胡萝卜素通路上相关基因的表达量上调幅度

Table 2 Up-regulation of relative expression levels of carotenoid biosynthetic genes in leaves of the control and transgenic lines

基因 gene	WT	OE-1	OE-2	基因 gene	WT	OE-1	OE-2	基因 gene	WT	OE-1	OE-2
NtPSY	1	2.12	2.70	NtLCYB2	1	2.55	1.41	NtCCD4-b	1	15.21	5.65
NtPDS	1	4.06	3.51	NtVDE	1	1.66	2.04	NtCCD4-c	1	5.83	5.98
NtZDS	1	4.20	3.27	NtNXC	1	0.92	2.42	NtNCED3-a	1	3.91	11.29
NtCRTISO	1	5.00	5.55	NtCCD1-a	1	0.65	7.16	NtNCED3-b	1	26.54	21.82
NtLCYE	1	5.79	3.31	NtCCD1-b	1	1.17	0.52	NtNCED5	1	3.03	2.16
NtLCYB1	1	1.02	2.02	NtCCD4-a	1	2.49	4.95

参考文献 34

[1]	Ahrazem O, Rubio-Moraga A, Trapero A, Gómez-Gómez L. 2012. Developmental and stress regulation of gene expression for a 9-cis-epoxycarotenoid dioxygenase,CstNCED,isolated from Crocus sativus stigmas. Journal of Experimental Botany, 63 (2):681-694. doi: 10.1093/jxb/err293 URL
[2]	Burbidge A, Grieve T M, Jackson A, Thompson A, McCarty D R, Taylor I B. 1999. Characterization of the ABA‐deficient tomato mutant notabilis and its relationship with maize Vp14. The Plant Journal, 17 (4):427-431. doi: 10.1046/j.1365-313X.1999.00386.x URL
[3]	Chen Yuan-ping, Yang Wen-yu. 2005. Determination of GA₃,IAA,ABA and ZT in dormant buds of Allium ovalifolium by HPLC. Journal of Sichuan Agricultural University, 23 (4):498-500. (in Chinese)
	陈远平, 杨文钰. 2005. 卵叶韭休眠芽中GA₃、IAA、ABA和ZT的高效液相色谱法测定. 四川农业大学学报, 23 (4):498-500.
[4]	Frank H A, Brudvig G W. 2004. Redox functions of carotenoids in photosynthesis. Biochemistry, 43 (27):8607-8615. pmid: 15236568
[5]	Fu C C, Han Y C, Fan Z Q, Chen J Y, Chen W X, Lu W J, Kuang J F. 2016. The papaya transcription factor CpNAC 1 modulates carotenoid biosynthesis through activating phytoene desaturase genes CpPDS2/4 during fruit ripening. Journal of Agricultural & Food Chemistry, 64 (27):5454-5463.
[6]	Fu Jian-xin, Zhang Chao, Wang Yi-guang, Zhao Hong-bo. 2016. Reference gene selection for quantitative real-time polymerase chain reaction(qRT-PCR) normalization in the gene expression of sweet osmanthus tissues. Journal of Zhejiang A & F University, 33 (5):727-733. (in Chinese)
	付建新, 张超, 王艺光, 赵宏波. 2016. 桂花组织基因表达中荧光定量PCR内参基因的筛选. 浙江农林大学学报, 33 (5):727-733.
[7]	Gao S, Gao J, Zhu X Y, Song Y, Li Z P, Ren G D, Zhou X, Kuai B. 2016. ABF2,ABF3,and ABF 4 promote ABA-mediated chlorophyll degradation and leaf senescence by transcriptional activation of chlorophyll catabolic genes and senescence-associated genes in Arabidopsis. Molecular Plant, 9 (9):1272-1285. doi: 10.1016/j.molp.2016.06.006 URL
[8]	Han Y J, Wang X H, Chen W C, Dong M F, Yuan W J, Liu X, Shang F. 2013. Differential expression of carotenoid-related genes determines diversified carotenoid coloration in flower petal of Osmanthus fragrans. Tree Genetics & Genomes, 10 (2):329-338.
[9]	Hermanns A S, Zhou X S, Xu Q, Tadmor Y, Li L. 2020. Carotenoid pigment accumulation in horticultural plants. Horticultural Plant Journal, 6 (6):343-360. doi: 10.1016/j.hpj.2020.10.002 URL
[10]	Hou Dan. 2014. Analysis of floral scent and pigment cons tituents and thereflectance to temperature fluctuation in Osmanthus fragrans[M. D. Dissertation]. Hangzhou: Zhejiang A & F University. (in Chinese)
	侯丹. 2014. 桂花主要品种花香和花色及其对温度变化的响应[硕士论文]. 杭州: 浙江农林大学.
[11]	Iuchi S, Kobayashi M, Taji T, Naramoto M, Seki M, Kato T, Tabata S, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K. 2001. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase,a key enzyme in abscisic acid biosynthesis in Arabidopsis. The Plant Journal:for Cell and Molecular Biology, 27 (4):325-333. doi: 10.1046/j.1365-313x.2001.01096.x URL
[12]	Jia L D, Wang J S, Wang R, Duan M Z, Qiao C L, Chen X, Ma G Q, Zhou X T, Zhu M C, Jing F Y. 2021. Comparative transcriptomic and metabolomic analyses of carotenoid biosynthesis reveal the basis of white petal color in Brassica napus. Planta, 253 (1):1-14. doi: 10.1007/s00425-020-03501-3
[13]	Lu S W, Zhang Y, Zhu K J, Yang W, Ye J L, Chai L J, Xu Q, Deng X X. 2018. The citrus transcription factor CsMADS 6 modulates carotenoid metabolism by directly regulating carotenogenic genes. Plant Physiology, 176 (4):2657-2676. doi: 10.1104/pp.17.01830 URL
[14]	Lu S W, Ye J L, Zhu K J, Zhang Y, Zhang M W, Xu Q, Deng X X. 2021. A fruit ripening-associated transcription factor CsMADS 5 positively regulates carotenoid biosynthesis in citrus. Journal of Experimental Botany, 72 (8):3028-3043. doi: 10.1093/jxb/erab045 URL
[15]	Moran R. 1982. Formulae for determination of chlorophyllous pigments extracted with N,N-dimethylformamide. Plant Physiology, 69 (6):1376-1381. doi: 10.1104/pp.69.6.1376 pmid: 16662407
[16]	Nambara E, Marion-Poll A. 2005. Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol, 56 (1):165-185. doi: 10.1146/arplant.2005.56.issue-1 URL
[17]	Ning G G, Xiao X, Lv H R, Li X, Zuo Y, Bao M Z. 2012. Shortening tobacco life cycle accelerates functional gene identification in genomic research. Plant Biology, 14 (6):934-943. doi: 10.1111/j.1438-8677.2012.00571.x pmid: 23107371
[18]	Schwartz S H, Tan B C, Gage D A, Zeevaart J A D, McCarty D R. 1997. Specific oxidative cleavage of carotenoids by VP 14 of maize. Science, 276 (5320):1872-1874. doi: 10.1126/science.276.5320.1872 pmid: 9188535
[19]	Shi Y M, Guo J G, Zhang W, Jin L F, Liu P P, Chen X, Li F, Wei P, Li Z F, Li W Z. 2015. Cloning of the lycopene β-cyclase gene in Nicotiana tabacum and its overexpression confers salt and drought tolerance. International Journal of Molecular Sciences, 16 (12):30438-30457. doi: 10.3390/ijms161226243 URL
[20]	Simpson K, Fuentes P, Quiroz-Iturra L F, Flores-Ortiz C, Contreras R, Handford M, Stange C. 2018. Unraveling the induction of phytoene synthase 2 expression by salt stress and abscisic acid in Daucus carota. Journal of Experimental Botany, 69 (16):4113-4126. doi: 10.1093/jxb/ery207 pmid: 29860511
[21]	Sun L, Yuan B, Zhang M, Wang L, Cui M M, Wang Q, Leng P. 2012. Fruit-specific RNAi mediated suppression of SlNCED1 increases both lycopene and β-carotene contents in tomato fruit. Journal of Experimental Botany, 63 (8):3097-3108. doi: 10.1093/jxb/ers026 URL
[22]	Sun T H, Yuan H, Cao H B, Yazdani M, Tadmor Y, Li L. 2018. Carotenoid metabolism in plants: the role of plastids. Molecular Plant, 11 (1):58-74. doi: S1674-2052(17)30273-3 pmid: 28958604
[23]	Wang Hua, Pan Qi, Ju Meng, Wang Zi-yu, Wang Wang-wei, Wang Rui-sheng, Zhu Li-wu. 2019. Cloning and expression of PbpNCED3 gene from 'Huangguan' pear. Journal of South China Agricultural University, 40 (3):82-88. (in Chinese)
	王华, 潘祺, 鞠萌, 汪紫萸, 汪王微, 王瑞生, 朱立武. 2019. 黄冠梨PbpNCED3基因的克隆与表达. 华南农业大学学报, 40 (3):82-88.
[24]	Wang Xiao-qing, Zhang Chao, Wang Yan-jie, Dong Li. 2012. lsolation and expression of 9-cis epoxycarotenoid dioxygenase gene in tree peony. Acta Horticulturae Sinica, 39 (10):2033-2044. (in Chinese)
	王晓庆, 张超, 王彦杰, 董丽. 2012. 牡丹NCED基因的克隆和表达分析. 园艺学报, 39 (10):2033-2044.
[25]	Wang Y G, Zhang C, Dong B, Fu J X, Hu S Q, Zhao H B. 2018. Carotenoid accumulation and its contribution to flower coloration of Osmanthus fragrans. Frontiers in Plant Science, 9:1499-1515. doi: 10.3389/fpls.2018.01499 URL
[26]	Xiang Qi-bai, Liu Yu-lian. 2008. An illustrated monograph of the Sweet Osmanthus variety in China. Hangzhou: Zhejiang Science & Technology Press:86-88. (in Chinese)
	向其柏, 刘玉莲. 2008. 中国桂花品种图志. 杭州: 浙江科学技术出版社:86-88.
[27]	Yang M M, Zhu S B, Jiao B Z, Duan M, Meng Q W, Ma N, Lv W. 2020. SlSGRL,a tomato SGR-like protein,promotes chlorophyll degradation downstream of the ABA signaling pathway. Plant Physiology and Biochemistry, 157:316-327. doi: 10.1016/j.plaphy.2020.10.028 URL
[28]	Yuan H, Zhang J X, Nageswaran D, Li L. 2015. Carotenoid metabolism and regulation in horticultural crops. Horticulture Research, 2 (1):15036-15046. doi: 10.1038/hortres.2015.36
[29]	Zeng Xiang-ling. 2016. Research of TPS and CCD function analysis and their influence on petal color and scent in Osmanthus fragrans[Ph. D. Dissertation]. Wuhan: Huazhong Agricultural University. (in Chinese)
	曾祥玲. 2016. 桂花TPS和CCD功能分析及其对花瓣色香形成的影响研究[博士论文]. 武汉: 华中农业大学.
[30]	Zhang Chao, Luo Yun, Wang Yi-guang, Fu Jian-xin, Zhao Hong-bo. 2016. Cloning and expression analysis of 15-cis-ζ-carotene lsomerase gene(Z-ISO)in Osmanthus fragrans. Journal of Agricultural Biotechnology, 24 (10):1512-1521. (in Chinese)
	张超, 罗云, 王艺光, 付建新, 赵宏波. 2016. 桂花15-顺式-ζ-胡萝卜素异构酶基因(Z-ISO)的克隆及表达分析. 农业生物技术学报, 24 (10):1512-1521.
[31]	Zhang C, Wang Y G, Fu J X, Bao Z Y, Zhao H B. 2016a. Transcriptomic analysis and carotenogenic gene expression related to petal coloration in Osmanthus fragrans‘Yanhong Gui’. Trees-Structure and Function, 30 (4):1-17.
[32]	Zhang X S, Pei J J, Zhao L G, Tang F, Feng X Y, Xie J C. 2016b. Overexpression and characterization of CCD4 from Osmanthus fragrans and β-ionone biosynthesis from β-carotene in vitro. Journal of Molecular Catalysis B:Enzymatic, 134:105-114. doi: 10.1016/j.molcatb.2016.10.003 URL
[33]	Zhou Q Q, Li Q C, Li P, Zhang S T, Liu C, Jin J J, Cao P J, Yang Y X. 2019. Carotenoid cleavage dioxygenases: identification, expression, and evolutionary analysis of this gene family in tobacco. International Journal of Molecular Sciences, 20 (22):5796-5819. doi: 10.3390/ijms20225796 URL
[34]	Zhu K J, Sun Q, Chen H Y, Mei X H, Lu S W, Ye J L, Chai L J, Xu Q, Deng X X. 2021. Ethylene activation of carotenoid biosynthesis by a novel transcription factor CsERF061. Journal of Experimental Botany, 72 (8):3137-3154. doi: 10.1093/jxb/erab047 pmid: 33543285