‘西伯利亚’百合LiCMK基因克隆及功能分析

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

摘要/Abstract

摘要：

4-二磷酸胞苷-2-C-甲基赤藓糖激酶（CMK）是萜类花香合成甲基赤藓糖醇磷酸（MEP）途径的关键酶，为揭示其对百合花香的调控作用，从‘西伯利亚’百合（Lilium ‘Siberia’）中克隆LiCMK基因,进行生物信息学分析，利用亚细胞定位确定蛋白位置，采用荧光定量PCR技术检测基因时空表达模式，并运用病毒诱导基因沉默（VIGS）方法瞬时沉默LiCMK，验证其功能。结果显示，LiCMK全长1 200 bp，编码399个氨基酸，属于IspE家族，与油棕（Elaeis guineensis）中的CMK相似度最高。LiCMK的表达随花期呈现先上升后降低的规律，在盛花期的表达量达到峰值，在花器官中的表达量明显高于茎、叶中的表达量。LiCMK蛋白定位于叶绿体中。LiCMK在百合中瞬时沉默后，LiCMK表达水平下降了约84%，主要的单萜合成基因表达量明显下降，月桂烯合酶基因（MYS）、罗勒烯合酶基因（OCS）和芳樟醇合酶基因（LIS）分别降低了57%、59%和63%，主要的单萜类化合物月桂烯、罗勒烯和芳樟醇释放量显著下降。结果表明，LiCMK基因对‘西伯利亚’百合单萜化合物的合成与花香释放有重要作用，为后续深入研究百合单萜合成及其花香释放的调控机制奠定基础。

关键词: ‘西伯利亚’百合, 花香, LiCMK, 基因克隆, 表达分析, 基因沉默, 单萜化合物, 调控

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

刘思佳, 王浩楠, 付宇辰, 闫文欣, 胡增辉, 冷平生. ‘西伯利亚’百合LiCMK基因克隆及功能分析[J]. 生物技术通报, 2023, 39(3): 196-205.

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.

图/表 13

图1 ‘西伯利亚’百合花朵不同发育时期

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

图2 盛开期的‘西伯利亚’百合不同器官及组织

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

表1 引物序列

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

图3 ‘西伯利亚’百合LiCMK基因扩增产物电泳 M：DL2000 DNA marker；1：LiCMK扩增产物

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

图4 LiCMK蛋白与其他植物CMK同源性比对黑色：同源性=100%；灰色：同源性≥75%；浅灰色：同源性 ≥50%

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

图5 LiCMK蛋白与其他17种植物CMK蛋白的系统进化分析

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

图6 ‘西伯利亚’百合 LiCMK 蛋白质亲疏水性的预测

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

图7 ‘西伯利亚’百合LiCMK蛋白质二级结构的预测蓝色为α-螺旋，红色为延伸链，绿色为β-转角，紫色为无规卷曲

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

图8 ‘西伯利亚’百合LiCMK蛋白质三级结构的预测

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

图9 ‘西伯利亚’百合LiCMK在不同花期与不同组织的表达分析图中误差线表示标准偏差，不同小写字母表示差异显著P<0.05，下同

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

图10 ‘西伯利亚’百合 LiCMK亚细胞定位从左到右依次为目标蛋白荧光通道、明场、叶绿体荧光通道、叠加图

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

图11 野生型组与阴性对照组‘西伯利亚’百合花朵比较与LiCMK基因沉默效率

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

图12 ‘西伯利亚’百合中LiCMK基因沉默对单萜合酶基因表达（A）及单萜释放量的影响（B）

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

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