金银花光形态建成因子LjCOP1基因克隆及表达模式分析

doi:10.3969/j.issn.1004-1524.20230896

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (6): 1290-1299.DOI: 10.3969/j.issn.1004-1524.20230896

金银花光形态建成因子LjCOP1基因克隆及表达模式分析

宛燕¹^,²(), 周晓春³, 房海灵¹^,²^,^*(), 林沂¹, 亓希武², 于盱², 陈泽群², 梁呈元²^,^*()

1.南京中医药大学,江苏南京 210023
2.江苏省中国科学院植物研究所,江苏南京 210014
3.盐城市大丰区林场,江苏盐城 224100

收稿日期:2023-07-19 出版日期:2024-06-25 发布日期:2024-07-02
作者简介:宛燕(1998—),女,安徽巢湖人,硕士研究生,主要从事药用植物资源研究。E-mail: wany998061@163.com
通讯作者: *房海灵,E-mail: fanghailing2013@163.com;梁呈元,E-mail: chenling8410@126.com
基金资助:
国家自然科学基金(32000262);江苏省林业科技创新与推广项目(LYKJ〔2021〕20);常州市科技支撑计划项目(CE20202006)

Cloning of light regulator LjCOP1 in Lonicera japonica and its expression characteristics

WAN Yan¹^,²(), ZHOU Xiaochun³, FANG Hailing¹^,²^,^*(), LIN Yi¹, QI Xiwu², YU Xu², CHEN Zequn², LIANG Chengyuan²^,^*()

1. Nanjing University of Chinese Medicine, Nanjing 210023, China
2. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China
3. Yancheng Dafeng District Forest Farm, Yancheng 224100, Jiangsu, China

Received:2023-07-19 Online:2024-06-25 Published:2024-07-02

摘要/Abstract

摘要：

光是影响植物生长发育及次生代谢合成的最重要的环境因子之一,COP1 E3连接酶是光信号转导的分子开关。该研究基于金银花转录组数据库,从金银花(Lonicera japonica Thunb.)中克隆得到LjCOP1基因,并对其进行生物信息学及表达模式分析。结果表明,该基因包含13个外显子,编码区(CDS)长度为2 034 bp,编码677个氨基酸残基。多序列比对显示,LjCOP1与其他植物COP1序列具有较高保守性,均包含环形锌指结合域、卷曲螺旋形和WD40重复序列等特殊结构域。基于SWISS-MODEL对金银花LjCOP1基因进行的蛋白结构预测显示其与拟南芥AtCOP1在蛋白质三级结构层面高度保守。启动子顺式作用元件分析表明,LjCOP1启动子序列中含有大量激素响应元件、光响应元件和胁迫响应元件。系统发生分析表明,LjCOP1与莴笋LsCOP1基因亲缘关系较近。表达模式分析显示,LjCOP1在金银花不同部位及花发育时期均有表达,且在叶中高量表达。与黑暗条件相比,白光下LjCOP1基因表达丰度最高,且对黑暗转蓝光响应更强烈。

关键词: 金银花, LjCOP1基因, 表达模式

Abstract:

Light is one of the most important environmental factors to regulate growth, development and metabolism of plants, and COP1 (Constitutively photomorphogenic1) is a molecular switch for light signal transduction. In this study, LjCOP1 gene was cloned from honeysuckle (Lonicera japonica Thunb.) based on the honeysuckle transcriptome database, and analyzed for its bioinformatics and expression pattern. The results showed that LjCOP1 contains 13 exons and 12 introns, with a coding sequence (CDS) length of 2 034 bp encoding 677 amino acid residues. Multiple sequence comparison showed that LjCOP1 is highly conserved with other plant COP1 sequences, and all of them contain special structural domains such as ring zinc finger binding domain, coiled-coil domain and WD40 repeats. Protein structure prediction based on SWISS-MODEL showed that LjCOP1 was highly conserved with AtCOP1 of Arabidopsis thaliana at the level of protein tertiary structure. The analysis of promoter cis-acting progenitor showed that pLjCOP1 contained a large number of hormone-responsive, light-responsive, and stress-responsive elements. Phylogenetic analysis showed that LjCOP1 and LsCOP1 in lettuce genes were closely related. Furthermore, expression pattern analysis showed that LjCOP1 was expressed in different parts of honeysuckle and during the period of flower development, and was highly expressed in leaves. Compared with the dark condition, the highest gene abundance of LjCOP1 was found in white light, and responded more strongly to dark-to-blue light.

Key words: Lonicera japonica, LjCOP1 gene, expression characteristic

中图分类号:

S567.9

宛燕, 周晓春, 房海灵, 林沂, 亓希武, 于盱, 陈泽群, 梁呈元. 金银花光形态建成因子LjCOP1基因克隆及表达模式分析[J]. 浙江农业学报, 2024, 36(6): 1290-1299.

WAN Yan, ZHOU Xiaochun, FANG Hailing, LIN Yi, QI Xiwu, YU Xu, CHEN Zequn, LIANG Chengyuan. Cloning of light regulator LjCOP1 in Lonicera japonica and its expression characteristics[J]. Acta Agriculturae Zhejiangensis, 2024, 36(6): 1290-1299.

图/表 8

表1 LjCOP1基因克隆及表达所用引物

Table 1 Primers used for cloning and expression of LjCOP1 gene

引物名称 Primer name	序列 Primer sequence(5'-3')
LjCOP1-F	ATGGCTGCTGATAGTTCAGTTGGG
LjCOP1-R	TCATCTTTTCGTTGCGACAAAAAAG
LjCOP1pro-F	TATAAAGCCTTCAATCGCTAAG
LjCOP1pro-R	ATCCCTGTCCAACTCATCC
qLjCOP1-F	GAGAAACATAAGCCAACC
qLjCOP1-R	TATTCGCCATTTACCG
qLjActin-F	TGCTGGATTCTGGTGATGGT
qLjActin-R	ATTTCCCGCTCTGCTGTG

图1 LjCOP1基因PCR产物电泳结果(A)、基因结构(B)及不同植物COP1序列比对(C) B图中矩形为外显子,线条为内含子;C图中横线表示保守的RING、Coiled coil和WD40结构域。

Fig.1 Electrophoresis results (A) of LjCOP1 gene PCR products and gene structure (B), and comparison of COP1 sequences in different plants (C) Rectangles in Figure B are exons, lines are introns; The horizontal lines in Figure C represent conserved RING, coiled coil and WD40 domains.

图2 金银花LjCOP1蛋白二级结构预测橙色螺旋为α-螺旋,紫色箭头为延伸链,粉色直线为无规则卷曲。

Fig.2 Prediction of the secondary structure of LjCOP1 protein of L. japonica Orange helix is α-helix, purple arrow is extended strand, and pink line is random curl.

图3 金银花LjCOP1蛋白三级结构预测(A)以及金银花LjCOP1蛋白三级结构与拟南芥AtCOP1比对情况(B) 橙色片段为拟南芥COP1的蛋白结构,绿色片段为金银花LjCOP1蛋白SWISS-MODEL预测结果。

Fig.3 Prediction of tertiary structure of LjCOP1 protein in L. japonica(A) and comparison between the tertiary structure of LjCOP1 and AtCOP1 (B) The orange fragment is the protein structure of Arabidopsis COP1, and the green fragment is the SWISS-MODEL prediction result of LjCOP1 protein of L. japonica.

图4 金银花LjCOP1基因启动子及调控元件分析红色字体代表起始密码子,上游位置为负。

Fig.4 Analysis of promoter and regulatory elements of LjCOP1 gene in L. japonica The red font represents the start codon and the upstream position is negative.

图5 金银花LjCOP1系统进化分析

Fig.5 Phylogenetic analysis of the LjCOP1 in L. japonica

图6 LjCOP1基因在金银花中的时空表达分析 A, LjCOP1基因在金银花不同组织部位表达分析;B, LjCOP1基因在金银花不同花期表达分析,其中YA为幼蕾期, GA为三青期, WAI为二白期, WAII为大白期, SF为银花期, GF为金花期。不同处理间没有相同小写字母表示差异显著(P<0.05),下同。

Fig.6 Temporal and spatial expression analysis of LjCOP1 gene in L. japonica A, Expression analysis of LjCOP1 gene in different tissue parts of L. japonica;B, Expression analysis of LjCOP1 gene in different flowering stages of L. japonica,in Figure B, YA is the young alabastrum, GA is the green alabastrum, WAI is the white alabastrum I, WAII is the white alabastrum Ⅱ, SF is the silvery flower, and GF is the golden flower. The bars with different lowercase letters indicate the significant difference (P<0.05). The same as below.

图7 不同光照条件金银花LjCOP1基因表达分析 A, 黑暗;B,白光;C,红光;D,蓝光。

Fig.7 Analysis of LjCOP1 gene expression in L. japonica under different light conditions A, Dark; B, White light; C, Red light; D, Blue light.

参考文献 22

[1]	国家药典委员会. 中华人民共和国药典一部: 2020年版[M]. 北京: 中国医药科技出版社, 2020: 230.
[2]	LEE H J, SEO H S, RYU J, et al. Luteolin inhibited the gene expression, production and secretion of MUC5AC mucin via regulation of nuclear factor kappa B signaling pathway in human airway epithelial cells[J]. Pulmonary Pharmacology & Therapeutics, 2015, 31: 117-122.
[3]	WAN H Q, GE L L, LI J M, et al. Effects of a novel biflavonoid of Lonicera japonica flower buds on modulating apoptosis under different oxidative conditions in hepatoma cells[J]. Phytomedicine, 2019, 57: 282-291.
[4]	XIAO Y, LI B, LIU J. A new constituent against rheumatoid arthritis from the flower buds of Lonicera japonica[J]. Phytochemistry Letters, 2019, 29: 160-164.
[5]	LIU Q, FANG J P, WANG P P, et al. Characterization of a pectin from Lonicera japonica Thunb. and its inhibition effect on Aβ₄₂ aggregation and promotion of neuritogenesis[J]. International Journal of Biological Macromolecules, 2018, 107: 112-120.
[6]	KU S K, SEO B I, PARK J H, et al. Effect of Lonicerae Flos extracts on reflux esophagitis with antioxidant activity[J]. World Journal of Gastroenterology, 2009, 15(38): 4799-4805.
[7]	新型冠状病毒肺炎诊疗方案(试行第九版)[J]. 中国医药, 2022, 17(4): 481-487.
[8]	刘新, 王梦皎, 李一鸣, 等. 光强对金银花药材性状、光合特性及抗氧化酶活性的影响[J]. 江西农业学报, 2018, 30(9): 58-62.
	LIU X, WANG M J, LI Y M, et al. Effects of light intensity on medicinal characters, photosynthetic traits and antioxidative enzyme activities of Lonicera japonica[J]. Acta Agriculturae Jiangxi, 2018, 30(9): 58-62. (in Chinese with English abstract)
[9]	XU P B, ZAWORA C, LI Y, et al. Transcriptome sequencing reveals role of light in promoting anthocyanin accumulation of strawberry fruit[J]. Plant Growth Regulation, 2018, 86(1): 121-132.
[10]	AHMAD M, CASHMORE A R. The blue-light receptor cryptochrome 1 shows functional dependence on phytochrome A or phytochrome B in Arabidopsis thaliana[J]. The Plant Journal: for Cell and Molecular Biology, 1997, 11(3): 421-427.
[11]	DENG X W, CASPAR T, QUAIL P H. cop1: a regulatory locus involved in light-controlled development and gene expression in Arabidopsis[J]. Genes & Development, 1991, 5(7): 1172-1182.
[12]	MAIER A, SCHRADER A, KOKKELINK L, et al. Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis[J]. The Plant Journal, 2013, 74(4): 638-651.
[13]	XU D Q. COP1 and BBXs-HY5-mediated light signal transduction in plants[J]. The New Phytologist, 2020, 228(6): 1748-1753.
[14]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. The New Phytologist, 2001, 25(4): 402-408.
[15]	LAU O S, DENG X W. The photomorphogenic repressors COP1 and DET1: 20 years later[J]. Trends in Plant Science, 2012, 17(10): 584-593.
[16]	崔连花, 詹为民, 杨陆浩, 等. 2个玉米ZmCOP1基因的克隆及其转录丰度对不同光质处理的响应[J]. 作物学报, 2022, 48(6): 1312-1324.
	CUI L H, ZHAN W M, YANG L H, et al. Molecular cloning of two maize(Zea mays) ZmCOP1 genes and their transcription abundances in response to different light treatments[J]. Acta Agronomica Sinica, 2022, 48(6): 1312-1324. (in Chinese with English abstract)
[17]	杨永庆, 胡朋举, 李玉荣, 等. 花生COP1基因的克隆及表达特征分析[J/OL]. 中国油料作物学报: 1-7 [2023- 07-12]. DOI: 10.19802/j.issn.1007-9084.2022363
	YANG Y Q, HU P J, LI Y R, et al. Cloning and expression analysis of COP1 gene in peanut[J/OL]. Chinese Journal of Oil Crop Sciences: 1-7 [2023-07-12]. DOI: 10.19802/j.issn.1007-9084.2022363 (in Chinese)
[18]	QI X W, FANG H L, CHEN Z Q, et al. Ectopic expression of a R2R3-MYB transcription factor gene LjaMYB12 from Lonicera japonica increases flavonoid accumulation in Arabidopsis thaliana[J]. International Journal of Molecular Sciences, 2019, 20(18): 4494.
[19]	张彬, 路阳, 尹美强, 等. 紫甘蓝COP1基因的克隆及表达模式分析[J]. 山西农业大学学报(自然科学版), 2014, 34(5): 385-390.
	ZHANG B, LU Y, YIN M Q, et al. Cloning and expression pattern analysis of purple cabbage COP1 gene[J]. Journal of Shanxi Agricultural University(Natural Science Edition), 2014, 34(5): 385-390. (in Chinese with English abstract)
[20]	王丽娟, 赵佳利, 杨姣, 等. 苦荞COP1基因的生物信息学分析及参与花色苷合成研究[J]. 西南农业学报, 2022, 35(9): 1977-1985.
	WANG L J, ZHAO J L, YANG J, et al. Bioinformatics analysis of COP1 gene in Tartary buckwheat and its involvement in anthocyanin synthesis[J]. Southwest China Journal of Agricultural Sciences, 2022, 35(9): 1977-1985. (in Chinese with English abstract)
[21]	JIANG M M, REN L, LIAN H L, et al. Novel insight into the mechanism underlying light-controlled anthocyanin accumulation in eggplant (Solanum melongena L.)[J]. Plant Science, 2016, 249: 46-58.
[22]	TAO R Y, BAI S L, NI J B, et al. The blue light signal transduction pathway is involved in anthocyanin accumulation in ‘Red Zaosu’ pear[J]. Planta, 2018, 248(1): 37-48.

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金银花光形态建成因子LjCOP1基因克隆及表达模式分析

Cloning of light regulator LjCOP1 in Lonicera japonica and its expression characteristics

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