Cloning and Functional Analysis of LiCMK Gene in Lilium ‘Siberia’

doi:10.13560/j.cnki.biotech.bull.1985.2022-0714

Abstract

Abstract:

4-diphosphocytidyl-2-C-methyl-D-erythritol kinase（CMK）is a crucial enzyme for terpenoid synthesis in the methylerythritol phosphate（MEP）pathway. To uncover its regulating effect on the aroma of the lilies, gene LiCMK was cloned from Lilium ‘Siberia’ and analyzed by bioinformatics. Specifically, the protein was located by subcellular localization and spatial-temporal expression patterns of LiCMK gene were explored by means of fluorescence quantitative PCR. And the validation was performed by instantaneously silenced LiCMK through viral induced gene silencing（VIGS）. As a result, LiCMK was 1 200 bp in length and encoded 399 amino acids, belonging to IspE family, which was relatively similar to CMK in Elaeis guineensis. The expression of LiCMK rose and fell corresponding to the flowering and reached the peak when it came to full flowering stage. Moreover, the expression in flower was significantly higher than that in stems and leaves. In addition, LiCMK protein was located in chloroplast. LiCMK expression dropped by about 84% after LiCMK was silenced instantaneously in Lilium ‘Siberia’, leading to the expressions of major monoterpene synthesis genes decreased, i.e., myrcene synthase gene（MYS）, ocimene synthase gene（OCS）and linalool synthase gene（LIS）plunged by 57%, 59% and 63%, respectively, and the release of major monoterpenes, myrcene, ocimene and linalool significantly decreased. To conclude, LiCMK gene is indispensable to the synthesis of monoterpenoid compounds and floral release in Lilium ‘Siberia’, which lays the foundation for future researches about the synthesis and regulatory mechanism of monoterpenoid compounds and the floral scent release.

Key words: Lilium ‘Siberia’, flower scent, LiCMK, gene cloning, expression analysis, VIGS, monoterpenes, regulate

LIU Si-jia, WANG Hao-nan, FU Yu-chen, YAN Wen-xin, HU Zeng-hui, LENG Ping-sheng. Cloning and Functional Analysis of LiCMK Gene in Lilium ‘Siberia’[J]. Biotechnology Bulletin, 2023, 39(3): 196-205.

Figures/Tables 13

Fig. 1 Different developmental stages of Lilium ‘Siberia’ flowers

Fig. 2 Different organs and tissues of Lilium ‘Siberia’ in full opening stage

Table1 Primer sequences

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
LiCMK-F	ATGGCCTCCTCTCACCT
LiCMK-R	TTATTCGGTTGCTGCTGCTG
qLiCMK-F	CCGATAACTTCTTCTGGATTCATCT
qLiCMK-R	GCCTGACCATTCTTGTAACTCTT
qLiLIS-F	TCGCCTTGTTGAGATCATCCTCTTC
qLiLIS-R	TGTCTCCACGGAGCTGCTGTT
qLiOCS-F	TCCGCCAATTACCACCCAACCA
qLiOCS-R	GCTGAGCCACTGGATCGTCTGA
qLiMYS-F	GACTTGTTGGAGCCATGTCTTAGAGA
qLiMYS-R	AACTCAACTCGTCGCCAAGAAGT
β-Actin-F	CGGTGTCTGGATTGGAGGGTCA
β-Actin-R	TTCGCTTTAGGACTTCGGGT
LiCMK-SF	AGTGGTCTCTGTCCAGTCCTATGGCCTCCTCTCACCT
LiCMK-SR	GGTCTCAGCAGACCACAAGTTTCGGTTGCTGCTGCTG
LiCMK-TF	AGTGGTCTCTGTCCAGTCCTATGGCCTCCTCTCACCT
LiCMK-TR	GGTCTCAGCAGACCACAAGTCAACCCAGCCTGCTTATCC

Fig. 3 Electrophoresis of amplified products of LiCMK gene in Lilium ‘Siberia’ M: DL2000 DNA marker. 1: Amplified products of LiCMK gene

Fig. 4 Homology comparison between LiCMK and CMK in other plants Black: The homology=100%. Grayness: The homology≥75%. Light grey: The homology≥50%

Fig. 5 Phylogenetic analysis of LiCMK proteins and CMK proteins of other 17 plants

Fig. 6 Prediction of hydrophilicity/hydrophobicity of LiCMK in Lilium ‘Siberia’

Fig. 7 Prediction of secondary structure of LiCMK protein in Lilium ‘Siberia’ The blue is α-helix, red is extended strand, green is beta turn, and purple is random coil

Fig. 8 Prediction of tertiary structure of LiCMK protein in Lilium ‘Siberia’

Fig. 9 Expression analysis of LiCMK in Lilium ‘Siberia’ at different flowering stages and LiCMK in different tissues The error line in the figure refers to the standard deviation. Different lowercase letters indicate significantly different at P<0.05. The same below

Fig. 10 Subcellular localization of LiCMK protein in Lilium ‘Siberia’ There are the target protein fluorescence channel, bright field, chloroplast fluorescence channel and merged one from left to right

Fig. 11 Comparison of Lilium ‘Siberia’ flowers between WT and pTRV2 groups and the silencing efficiency of gene LiCMK

Fig. 12 Effects of LiCMK silencing on the monoterpene synthase gene expression（A）and monoterpene release（B）in Lilium ‘Siberia’

References 37

[1]	孔滢, 等. 花香代谢与调控研究进展[J]. 北京林业大学学报, 2012, 34(2): 146-154.
	Kong Y, et al. Advances in metabolism and regulation of floral scent[J]. J Beijing For Univ, 2012, 34(2): 146-154.
[2]	Majetic CJ, Raguso RA, Ashman TL. The sweet smell of success: floral scent affects pollinator attraction and seed fitness in Hesperis matronalis[J]. Funct Ecol, 2009, 23(3): 480-487. doi: 10.1111/fec.2009.23.issue-3 URL
[3]	Unsicker SB, Kunert G, Gershenzon J. Protective perfumes: the role of vegetative volatiles in plant defense against herbivores[J]. Curr Opin Plant Biol, 2009, 12(4): 479-485. doi: 10.1016/j.pbi.2009.04.001 pmid: 19467919
[4]	孙丽超, 李淑英, 王凤忠, 等. 萜类化合物的合成生物学研究进展[J]. 生物技术通报, 2017, 33(1): 64-75. doi: 10.13560/j.cnki.biotech.bull.1985.2017.01.007
	Sun LC, Li SY, Wang FZ, et al. Research progresses in the synthetic biology of terpenoids[J]. Biotechnol Bull, 2017, 33(1): 64-75. doi: 10.13560/j.cnki.biotech.bull.1985.2017.01.007
[5]	Abbas F, Ke YG, Yu RC, et al. Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering[J]. Planta, 2017, 246(5): 803-816. doi: 10.1007/s00425-017-2749-x pmid: 28803364
[6]	Ramya M, An HR, Baek YS, et al. Orchid floral volatiles: Biosynthesis genes and transcriptional regulations[J]. Sci Hortic, 2018, 235: 62-69. doi: 10.1016/j.scienta.2017.12.049 URL
[7]	徐瑾, 等. 菊花不同花期及花序不同部位香气成分和挥发研究[J]. 西北植物学报, 2012, 32(4): 722-730.
	Xu J, et al. Studies of aroma compounds in Chrysanthemum in different florescence and inflorescence parts and aroma releasing[J]. Acta Bot Boreali Occidentalia Sin, 2012, 32(4): 722-730.
[8]	夏科, 蒋柏生, 赵志国, 等. 桂林地区不同桂花品种花香成分比较分析[J]. 广西植物, 2018, 38(11): 1493-1504.
	Xia K, Jiang BS, Zhao ZG, et al. Comparative analysis of aromatic components from different cultivars of Osmanthus fragrans in Guilin[J]. Guihaia, 2018, 38(11): 1493-1504.
[9]	Feng LG, Chen C, Li TL, et al. Flowery odor formation revealed by differential expression of monoterpene biosynthetic genes and monoterpene accumulation in rose(Rosa rugosa Thunb.)[J]. Plant Physiol Biochem, 2014, 75: 80-88. doi: 10.1016/j.plaphy.2013.12.006 URL
[10]	Yue YC, Yu RC, Fan YP. Transcriptome profiling provides new insights into the formation of floral scent in Hedychium coronari-um[J]. BMC Genomics, 2015, 16(1): 470. doi: 10.1186/s12864-015-1653-7 URL
[11]	Lee GW, Chung MS, Lee SS, et al. Transcriptome-guided identification and functional characterization of key terpene synthases involved in constitutive and methyl jasmonate-inducible volatile terpene formation in Eremochloa ophiuroides(Munro)Hack[J]. Plant Physiol Biochem, 2019, 141: 193-201. doi: 10.1016/j.plaphy.2019.05.032 URL
[12]	Du F, Wang T, Fan JM, et al. Volatile composition and classification of Lilium flower aroma types and identification, polymorphisms, and alternative splicing of their monoterpene synthase genes[J]. Hortic Res, 2019, 6: 110. doi: 10.1038/s41438-019-0192-9
[13]	郑冉冉, 吴景芝, 谷志佳, 等. 玫瑰香味玫红百合和橙香味紫红花滇百合的花香成分研究[J]. 浙江大学学报: 农业与生命科学版, 2021, 47(1): 32-42.
	Zheng RR, Wu JZ, Gu ZJ, et al. Study on the floral scent components of Lilium amoenum with rose fragrance and Lilium baker-ianum var. rubrum with orange fragrance[J]. J Zhejiang Univ Agric Life Sci, 2021, 47(1): 32-42.
[14]	Hu ZH, Tang B, Wu Q, et al. Transcriptome sequencing analysis reveals a difference in monoterpene biosynthesis between scented Lilium ‘Siberia’ and unscented Lilium ‘Novano’[J]. Front Plant Sci, 2017, 8: 1351. doi: 10.3389/fpls.2017.01351 URL
[15]	Muhlemann JK, et al. Floral volatiles: from biosynthesis to function[J]. Plant Cell Environ, 2014, 37(8): 1936-1949. doi: 10.1111/pce.12314 URL
[16]	Rohdich F, Lauw S, Kaiser J, et al. Isoprenoid biosynthesis in plants - 2C-methyl-D-erythritol-4-phosphate synthase(IspC protein)of Arabidopsis thaliana[J]. FEBS J, 2006, 273(19): 4446-4458. pmid: 16972937
[17]	Dudareva N, et al. (E)-β-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily[J]. Plant Cell, 2003, 15(5): 1227-1241. doi: 10.1105/tpc.011015 pmid: 12724546
[18]	Orlova I, Nagegowda DA, Kish CM, et al. The small subunit of snapdragon geranyl diphosphate synthase modifies the chain length specificity of tobacco geranylgeranyl diphosphate synthase in planta[J]. Plant Cell, 2009, 21(12): 4002-4017. doi: 10.1105/tpc.109.071282 URL
[19]	Dudareva N, Cseke L, Blanc VM, et al. Evolution of floral scent in Clarkia: novel patterns of S-linalool synthase gene expression in the C. breweri flower[J]. Plant Cell, 1996, 8(7): 1137-1148. doi: 10.1105/tpc.8.7.1137 pmid: 8768373
[20]	Gao FZ, et al. Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in flowers of Freesia × hybrida[J]. J Exp Bot, 2018, 69(18): 4249-4265. doi: 10.1093/jxb/ery224 URL
[21]	Zhang TX, Sun M, Guo YH, et al. Overexpression of LiDXS and LiDXR from lily(Lilium ‘Siberia’)enhances the terpenoid content in tobacco flowers[J]. Front Plant Sci, 2018, 9: 909. doi: 10.3389/fpls.2018.00909 URL
[22]	马波, 等. ‘西伯利亚’百合LiMCT基因的克隆及表达特性分析[J]. 分子植物育种, 2020, 18(18): 5933-5942.
	Ma B, et al. Cloning and expression characteristic analysis of LiMCT gene in Lilium ‘Siberia’[J]. Mol Plant Breed, 2020, 18(18): 5933-5942.
[23]	张茜, 罗景琳, 王浩楠, 等. 百合花香合成相关基因LiMCS的克隆、定位和表达特性研究[J]. 西北农业学报, 2022, 31(8):981-989.
	Zhang X, Luo JL, Wang HN, et al. Cloning, localization and expression characteristics of flower fragrance synthesis related gene LiMCS in Lilium[J]. Acta Agric Boreali Occidentalis Sin, 2022, 31(8):981-989.
[24]	Zhang TX, Guo YH, Shi XJ, et al. Overexpression of LiTPS2 from a cultivar of lily(Lilium ‘Siberia’)enhances the monoterpenoids content in tobacco flowers[J]. Plant Physiol Biochem, 2020, 151: 391-399. doi: 10.1016/j.plaphy.2020.03.048 URL
[25]	Rohdich F, et al. Biosynthesis of terpenoids: 4-diphosphocytidyl-2C-methyl-D-erythritol synthase of Arabidopsis thaliana[J]. Proc Natl Acad Sci USA, 2000, 97(12): 6451-6456. doi: 10.1073/pnas.97.12.6451 pmid: 10841550
[26]	Ahn CS, Pai HS. Physiological function of IspE, a plastid MEP pathway gene for isoprenoid biosynthesis, in organelle biogenesis and cell morphogenesis in Nicotiana benthamiana[J]. Plant Mol Biol, 2008, 66(5): 503-517. doi: 10.1007/s11103-007-9286-0 URL
[27]	乔洋洋. 盾叶薯蓣CMK基因和HDS基因的克隆与功能验证[D]. 武汉: 湖北大学, 2017.
	Qiao YY. Fuctional cloning of CMK gene and HDS gene from Dio-scorea zingiberensis C.H wright[D]. Wuhan: Hubei University, 2017.
[28]	范雨芳, 张曼, 向礼恩, 等. 黄花蒿CMK基因的克隆与功能分析[J]. 中国中药杂志, 2018, 43(11): 2254-2260.
	Fan YF, Zhang M, Xiang LE, et al. Molecular cloning and characterization of CMK from Artemisia annua[J]. China J Chin Mater Med, 2018, 43(11): 2254-2260.
[29]	汪周勇, 等. 金银花类药用植物IspE和IspH基因克隆和生物信息学分析[J]. 中国中药杂志, 2013, 38(1): 32-36.
	Wang ZY, et al. Cloning and bioinformatic analysis of IspE and IspH genes in Lonicera japonica and its substitutes[J]. China J Chin Mater Med, 2013, 38(1): 32-36.
[30]	Tang B, et al. Transcriptome analysis of the monoterpene biosynthesis pathway in petals of Lilium ‘Siberia’ at different flowering stages[J]. Plant Sci J, 2018, 36(2): 252-263.
[31]	张曼. 青蒿AaCMK、AaMCT、AaMCS基因的克隆与功能分析[D]. 重庆: 西南大学, 2016.
	Zhang M. Molecular cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene, 4-(cytidine 5'-diphospho)-2-C-methylery-thritol kinase gene, 2-C-methylerythritol-2, 4-cyclodiphosphate synthase gene from Artemisia annua L.[D]. Chongqing: Southwest University, 2016.
[32]	Gong YF, Liao ZH, Guo BH, et al. Molecular cloning and expression profile analysis of Ginkgo biloba DXS gene encoding 1-deoxy-D-xylulose 5-phosphate synthase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway[J]. Planta Med, 2006, 72(4): 329-335. doi: 10.1055/s-2005-916234 URL
[33]	Rohdich F, et al. Biosynthesis of terpenoids: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from tomato[J]. Proc Natl Acad Sci USA, 2000, 97(15): 8251-8256. doi: 10.1073/pnas.140209197 pmid: 10880567
[34]	童宇茹, 苏平, 赵瑜君, 等. 雷公藤4-(5'-二磷酸胞苷)-2-C-甲基-D-赤藓醇激酶基因的全长克隆与表达分析[J]. 中国中药杂志, 2015, 40(21): 4165-4170.
	Tong YR, Su P, Zhao YJ, et al. Cloning and expression analysis of 4-(cytidine-5-diphospho)-2-Cmethyl-D-erythritol kinase gene in Tripterygium wilfordii[J]. China J Chin Mater Med, 2015, 40(21): 4165-4170.
[35]	刘梦丽, 余函纹, 李景, 等. 桔梗中PgMCT和PgCMK的克隆及原核表达分析[J]. 中成药, 2022, 44(5): 1600-1605.
	Liu ML, Yu HW, Li J, et al. Cloning and prokaryotic expression analysis of PgMCT and PgCMK in Platycodon grandiflorus[J]. Chin Tradit Pat Med, 2022, 44(5): 1600-1605.
[36]	袁媛, 孙叶, 等. 植物花香代谢和基因工程研究进展[J]. 南方园艺, 2017, 28(5): 48-52.
	Yuan Y, Sun Y, et al. Advances in floral metabolism and gene engineering in plants[J]. South Hortic, 2017, 28(5): 48-52.
[37]	王淋, 等. 杜仲MVA和MEP途径相关基因的亚细胞定位与表达分析[J]. 植物研究, 2017, 37(1): 52-62. doi: 10.7525/j.issn.1673-5102.2017.01.008
	Wang L, et al. Subcellular localization and expression analysis of genes from Eucommia ulmoides involved in MVA and MEP pathway[J]. Bull Bot Res, 2017, 37(1): 52-62.