转录组与代谢组分析不同生长时期金线莲类胡萝卜素的积累

doi:10.13560/j.cnki.biotech.bull.1985.2024-0408

摘要/Abstract

摘要：

【目的】探究不同生长时期金线莲叶片中的类胡萝卜素的变化规律，解析其类胡萝卜素积累分子机制。【方法】选取不同生长时期（三月龄S3，六月龄S6，十月龄S10）金线莲叶片，采用液相色谱-质谱联用技术（LC-MS）和高通量转录组测序（RNA-seq）对不同生长时期的金线莲叶片进行分析。【结果】代谢组学结果表明参与叶片中类胡萝卜素合成的差异代谢物有23个，包括新黄质素、葡萄黄素、叶黄素和卡洛黄素等，转录组学结果表明差异代谢酶基因有21个，包括CYP97C1、CCD8、NCED、ZEP、PDS等，预测了ERF、MYB、C2H2、WRKY和bZIP等5种转录因子参与调控金线莲叶片类胡萝卜素的合成；并且进行了实时荧光定量PCR验证（RT-qPCR），实验表明选取的8个参与类胡萝卜素合成的酶基因在不同时期中的表达趋势与转录组学测序结果一致。【结论】金线莲不同生长时期ERF、MYB、C2H2等5种转录因子参与类胡萝卜素合成调控，与CYP97C1、CCD、NCED等5种关键差异酶基因关联明显，他们共同调控脱落酸醛、玉米黄质和叶黄素等6种关键差异代谢物。

关键词: 金线莲, 类胡萝卜素, 转录组, 代谢组, 不同时期

Abstract:

【Objective】 To investigate the variation rule of carotenoids in the leaves of Anoectochilus roxburghii at different growth stages, and to analyze the molecular mechanism of carotenoid accumulation.【Method】 The carotenoids contents in the leaves of A. roxburghii at different growth stages（S3, in three months old; S6, in six months old; and S10, in ten months old）were investigated by liquid chromatograph mass spectrometer（LC-MS）and high-throughput transcriptome sequencing（RNA-seq）.【Result】 Combining the transcriptome and metabolome to analyze the leaves of A. roxburghii at different growth periods, the metabolomics results showed that there were 23 differential metabolites involved in carotenoid synthesis in the leaves, including neoxanthin, graphemexanthin, luteolin, and caroxanthin, etc., and the transcriptome results showed that there were 21 differential metabolism enzyme genes, including CYP97C1, CCD8, NCED, ZEP, and PDS, etc., which predicted that 14 transcription factors, including ERF, MYB, C2H2, WRKY and bZIP, were involved in regulating the differential carotenoid synthesis and accumulation in A. roxburghii leaves. and real-time quantitative PCR（RT-qPCR）experiments were performed to show that the expression trends of eight selected enzyme genes involved in the synthesis of carotenoids in the different phases of the process were consistent with the results of the sequencing of transcriptomics.【Conclusion】 Five transcription factors, including ERF, MYB, and C2H2, were involved in the regulation of carotenoid synthesis during different growth periods of Anoectochilus roxburghii, and were significantly associated with five key differential enzyme genes, including CYP97C1, CCD, and NCED, which together regulate six key differential metabolites, including Abscisic aldehyde, Zeaxanthin, and Lutein.

Key words: Anoectochilus roxburghii, carotenoids, transcriptome, metabolome, different stages

梁婉凤, 曾菁菁, 胡若群, 曹佳玉, 郑涛, 李銮, 仇明月, 梁晓英, 陈莹. 转录组与代谢组分析不同生长时期金线莲类胡萝卜素的积累[J]. 生物技术通报, 2024, 40(10): 262-274.

LIANG Wan-feng, ZENG Jing-jing, HU Ruo-qun, CAO Jia-yu, ZHENG Tao, LI Luan, QIU Ming-yue, LIANG Xiao-ying, CHEN Ying. Transcriptional and Metabolomic Analysis of Carotenoid Accumulation in Anoectochilus roxburghii during Different Growth Periods[J]. Biotechnology Bulletin, 2024, 40(10): 262-274.

图/表 12

图1 不同生长阶段的金线莲从左到右依次为3月龄、6月龄和10月龄。下同

Fig. 1 A. roxburghii at different growth stages 3-month（S3）, 6-month（S6）and 10-month（S10）from left to right. The same below

表1 RT-qPCR引物

Table 1 Primers for RT-qPCR

Gene	Gene ID	Primer sequence（5'-3'）	Primer sequence（3'-5'）
Actin	Reference gene	CTGTTTGCAGCCCAAGACAC	CAGCCTCCACAGCTTCAAGA
crtISO	TRINITY_DN4193_c0_g1	TGGGAAGGACTTGACCGTCGTAG	ACCAACGCCACATCACACTTCTC
CYP97C1	TRINITY_DN4271_c0_g1	TCTCCATGATCCGACTGCAC	GACCGAAAGTCGAGAGTCTCA
DWARF27	TRINITY_DN7678_c0_g1	GCCTTGCTATGCCAACCTTT	TGCTGGGATTGGCCAAAGAA
crtB	TRINITY_DN28838_c0_g1	TAGTGGGTGCAGGTTTCAGC	AATGGCTACATCTCCTGCCG
CCD8	TRINITY_DN5697_c0_g1	AGATTGCCGCACACCGAGTTG	AGCTCACCCTCCCATCGTTCTC
crtZ	TRINITY_DN67725_c0_g2	AAGGCGCCAACACAGAATTG	CATGATTGCCGCCACCAAAT
ABA2	TRINITY_DN8894_c0_g1	GGTGTCACTGGCTCGAAAGT	GACACGCTCGCAAGAGAGAT
ZEP	TRINITY_DN9811_c0_g1	TACCCGCACTTTAGGGAACG	CGCTAACACCAGTCCACCAA

图2 样本主成分分析图图中同一颜色为一个组别，数字标号指同组的不同重复，如S3-3是3月龄组别的第3个重复，下同

Fig. 2 Principal component analysis plot for samples The same color in the figure is a group, and the numerical labels refer to different replicates of the same group, e.g., S3-3 is the third replicate of the 3-month group, the same below

图3 样本相关性热图图上蓝色、绿色、粉色代表的是分别是3、6、10月龄，框内蓝色颜色越深相关性越深

Fig. 3 Heat map of sample correlation The blue, green, and pink colors on the chart represent 3-, 6-, and 10-month, respectively; the darker the blue color in the box, the deeper the correlation

表2 不同生长时期金线莲叶片中差异类胡萝卜素代谢物

Table 2 Differential carotenoid metabolites in the leaves of A. roxburghii at different growth stages

代谢物 Metabolite	S3_vs_S6 regulated	S6_vs_S10 regulated	S3_vs_S10 regulated	分子式Formula	KEGG注释 KEGG annotation
1'-羟基-gamma-胡萝卜素葡萄糖苷 1'-Hydroxy-gamma-carotene glucoside	Unchanged	Up	Unchanged	C₄₆H₆₈O₆	C15861
脱落酸葡萄糖酯Abscisic acid glucose ester	Up	Unchanged	Unchanged	C₂₁H₃₀O₉	C15970
酮基R.g.-Keto III	Up	Unchanged	Up	C₄₂H₆₀O₄	C16278
辣椒素Capsorubin	Unchanged	Unchanged	Down	C₄₀H₅₆O₄	C08585
羟基螺旋黄质Hydroxyspirilloxanthin	Down	Unchanged	Down	C₄₁H₅₈O₂	C15879
新黄质素Neoxanthin	Up	Unchanged	Up	C₄₀H₅₆O₄	C08606
葡萄黄素Staphyloxanthin	Unchanged	Up	Up	C₅₁H₇₈O₈	C16148
诺吐黄素Nostoxanthin	Unchanged	Up	Up	C₄₀H₅₆O₄	C16284
2-酮基螺旋体黄质2-Ketospirilloxanthin	Unchanged	Unchanged	Down	C₄₂H₅₈O₃	C15884
羟基氯巴坦Hydroxychlorobactene	Down	Up	Unchanged	C₄₀H₅₄O	C15911
3-羟基叶茶烯酮3-Hydroxyechinenone	Unchanged	Up	Unchanged	C₄₀H₅₄O₂	C15966
三苯甲内酯 ABC 环Strigolactone ABC-rings	Down	Down	Down	C₁₄H₁₈O₃	C18036
8'-羟基琥珀酸酯8'-Hydroxyabscisate	Unchanged	Up	Up	C₁₅H₂₀O₅	C15514
脱落酸醛Abscisic aldehyde	Down	Unchanged	Down	C₁₅H₂₀O₃	C13455
羟基氯巴坦葡萄糖苷Hydroxychlorobactene glucoside	Down	Unchanged	Down	C₄₆H₆₄O₆	C15914
（2'S）-脱氧木酚 2'-alpha-L-岩藻糖苷（2'S）-Deoxymyxol 2'-alpha-L-fucoside	Down	Unchanged	Down	C₄₆H₆₆O₆	C15940
（2'S）-脱氧木酚 2'-（2,4-二-O-甲基-alpha-L-岩藻糖苷）（2'S）-Deoxymyxol 2'-（2,4-di-O-methyl-alpha-L-fucoside）	Up	Down	Unchanged	C₄₈H₇₀O₆	C15935
（3R,2'S）-2'-（2,4-二-O-甲基-alpha-L-岩藻糖苷）岩藻醇（3R,2'S）-Myxol 2'-（2,4-di-O-methyl-alpha-L-fucoside）	Down	Up	Unchanged	C₄₈H₇₀O₇	C15937
腓尼基黄质Phoenicoxanthin	Up	Unchanged	Up	C₄₀H₅₂O₃	C15967
玉米黄质Zeinoxanthin	Unchanged	Up	Unchanged	C₄₀H₅₆O	C08590
腺嘌呤Adonixanthin	Up	Unchanged	Up	C₄₀H₅₄O₃	C15968
叶黄素Lutein	Up	Unchanged	Up	C₄₀H₅₆O₂	C08601
卡洛黄素Caloxanthin	Up	Unchanged	Unchanged	C₄₀H₅₆O₃	C16282

表2 不同生长时期金线莲叶片中差异类胡萝卜素代谢物

Table 2 Differential carotenoid metabolites in the leaves of A. roxburghii at different growth stages

代谢物 Metabolite	S3_vs_S6 regulated	S6_vs_S10 regulated	S3_vs_S10 regulated	分子式Formula	KEGG注释 KEGG annotation
1'-羟基-gamma-胡萝卜素葡萄糖苷 1'-Hydroxy-gamma-carotene glucoside	Unchanged	Up	Unchanged	C₄₆H₆₈O₆	C15861
脱落酸葡萄糖酯Abscisic acid glucose ester	Up	Unchanged	Unchanged	C₂₁H₃₀O₉	C15970
酮基R.g.-Keto III	Up	Unchanged	Up	C₄₂H₆₀O₄	C16278
辣椒素Capsorubin	Unchanged	Unchanged	Down	C₄₀H₅₆O₄	C08585
羟基螺旋黄质Hydroxyspirilloxanthin	Down	Unchanged	Down	C₄₁H₅₈O₂	C15879
新黄质素Neoxanthin	Up	Unchanged	Up	C₄₀H₅₆O₄	C08606
葡萄黄素Staphyloxanthin	Unchanged	Up	Up	C₅₁H₇₈O₈	C16148
诺吐黄素Nostoxanthin	Unchanged	Up	Up	C₄₀H₅₆O₄	C16284
2-酮基螺旋体黄质2-Ketospirilloxanthin	Unchanged	Unchanged	Down	C₄₂H₅₈O₃	C15884
羟基氯巴坦Hydroxychlorobactene	Down	Up	Unchanged	C₄₀H₅₄O	C15911
3-羟基叶茶烯酮3-Hydroxyechinenone	Unchanged	Up	Unchanged	C₄₀H₅₄O₂	C15966
三苯甲内酯 ABC 环Strigolactone ABC-rings	Down	Down	Down	C₁₄H₁₈O₃	C18036
8'-羟基琥珀酸酯8'-Hydroxyabscisate	Unchanged	Up	Up	C₁₅H₂₀O₅	C15514
脱落酸醛Abscisic aldehyde	Down	Unchanged	Down	C₁₅H₂₀O₃	C13455
羟基氯巴坦葡萄糖苷Hydroxychlorobactene glucoside	Down	Unchanged	Down	C₄₆H₆₄O₆	C15914
（2'S）-脱氧木酚 2'-alpha-L-岩藻糖苷（2'S）-Deoxymyxol 2'-alpha-L-fucoside	Down	Unchanged	Down	C₄₆H₆₆O₆	C15940
（2'S）-脱氧木酚 2'-（2,4-二-O-甲基-alpha-L-岩藻糖苷）（2'S）-Deoxymyxol 2'-（2,4-di-O-methyl-alpha-L-fucoside）	Up	Down	Unchanged	C₄₈H₇₀O₆	C15935
（3R,2'S）-2'-（2,4-二-O-甲基-alpha-L-岩藻糖苷）岩藻醇（3R,2'S）-Myxol 2'-（2,4-di-O-methyl-alpha-L-fucoside）	Down	Up	Unchanged	C₄₈H₇₀O₇	C15937
腓尼基黄质Phoenicoxanthin	Up	Unchanged	Up	C₄₀H₅₂O₃	C15967
玉米黄质Zeinoxanthin	Unchanged	Up	Unchanged	C₄₀H₅₆O	C08590
腺嘌呤Adonixanthin	Up	Unchanged	Up	C₄₀H₅₄O₃	C15968
叶黄素Lutein	Up	Unchanged	Up	C₄₀H₅₆O₂	C08601
卡洛黄素Caloxanthin	Up	Unchanged	Unchanged	C₄₀H₅₆O₃	C16282

图4 不同生长时期金线莲叶片差异类胡萝卜素物质代谢物聚类热图

Fig. 4 Clustering heat map of metabolites of differential carotenoid substances in the leaves of A. roxburghii at different growth stages

图5 不同生长时期金线莲叶片差异类胡萝卜素物质代谢物变化比例图浅黄色柱状代表3月龄和6月龄的比较，粉色柱状代表3月龄和10月龄的比较，棕色柱状代表6月龄和10月龄的比较，柱状越长变化越大，框内数字代表变化倍数

Fig. 5 Proportion of changes in the metabolites of different carotenoids in the leaves of A. roxburghii at different growth stages Light yellow bars indicate the comparison between 3-month and 6-month, pink bars indicate the comparison between 3-month and 10-month, and brown bars indicate the comparison between 6-month and 10-month. The longer the bar, the greater the change, and the numbers in the box indicate multiples of the change

表3 不同生长时期金线莲叶片中差异类胡萝卜素酶基因

Table 3 Differential carotenoid enzyme genes in A. roxburghii leaves at different growth stages

基因 Gene	ID	S3 VS S6 DESeq regulated	S6 VS S10 DESeq regulated	S3 VS S10 DESeq regulated	对应酶 Corresponding enzyme	酶KO Enzyme KO
CCD8	TRINITY_DN1716_c0_g1	Down	Normal	Normal	[EC:1.13.11.68]	K17912
	TRINITY_DN5697_c0_g1	Down	Normal	Down	[EC:1.13.11.69 1.13.11.70]	K17913
	TRINITY_DN8190_c0_g1	Down	Normal	Down	[EC:1.13.11.69 1.13.11.70]	K02293
PDS	TRINITY_DN9_c1_g1	Normal	Normal	Up	[EC:1.3.5.5]	K02293
NCED	TRINITY_DN11998_c0_g1	Up	Normal	Up	[EC:1.13.11.51]	K09840
NCED	TRINITY_DN9005_c0_g1	Up	Up	Up	[EC:1.13.11.51]	K15746
crtISO	TRINITY_DN4193_c0_g1	Up	Normal	Normal	[EC:5.2.1.13]	K09835
crtISO	TRINITY_DN1162_c2_g1	Down	Normal	Normal	[EC:5.2.1.13]	K09843
crtZ	TRINITY_DN6176_c0_g1	Up	Normal	Normal	[EC:1.14.15.24]	K15746
crtZ	TRINITY_DN67725_c0_g2	Down	Up	Normal	[EC:1.14.15.24]	K09838
crtB	TRINITY_DN28838_c0_g1	Up	Up	Normal	[EC:2.5.1.32]	K02291
crtB	TRINITY_DN4528_c0_g1	Down	Down	Normal	[EC:2.5.1.32]	K09841
CYP97C1	TRINITY_DN5659_c0_g1	Down	Normal	Down	[EC:1.14.14.137]	K09843
	TRINITY_DN4271_c0_g1	Down	Normal	Down	[EC:1.14.14.137]	K14595
	TRINITY_DN586_c0_g1	Down	Down	Normal	[EC:1.14.14.158]	K09837
ZEP	TRINITY_DN9811_c0_g1	Up	Normal	Normal	[EC:1.14.15.21]	K09838
LUT5, CYP97A3	TRINITY_DN2395_c0_g1	Up	Normal	Normal	[EC:1.14.15.21]	K02293
DWARF27	TRINITY_DN7678_c0_g1	Up	Up	Normal	[EC:5.2.1.14]	K17911
ABA2	TRINITY_DN8894_c0_g1	Down	Normal	Normal	[EC:1.1.1.28]	K09841
crtL2	TRINITY_DN7101_c0_g1	Up	Normal	Up	[EC:5.5.1.18]	K06444
AOG	TRINITY_DN13229_c0_g1	Down	Down	Normal	[EC:2.4.1.26]	K14595

图6 类胡萝卜素合成相关差异基因聚类热图热图中橙色代表高表达水平紫色代表低表达水平，颜色越深程度越深

Fig. 6 Heat map of carotenoid synthesis-related differential gene clustering In the heat map, orange indicates high expression level and purple indicates low expression level. The darker the color, the deeper the degree

图7 类胡萝卜素转录代谢联合分析图图中实线框和虚线框分别代表主要通路及非主要，橙色英文和绿色中文为差异基因和差异代谢物，其中红色蓝色代表着差异代谢物在热图中表达由高到低，而橙色紫色则代表着差异转录基因在热图中表达由高到低

Fig. 7 Co-analysis map of carotenoid transcript metabolism Solid and dashed boxes indicate major pathways and non-major, respectively; orange English and green Chinese are differential genes and differential metabolites, where red-blue indicates differential metabolites expressed from high to low in the heat map, and orange-purple indicates differential transcribed genes expressed from high to low in the heat map

图8 类胡萝卜素转录代谢中相关转录因子与酶基因调控关系图绿色图形代表酶基因，紫色图形代表转录因子，实线和虚线分别代表正负相关性，线条越粗相关性越强

Fig. 8 Regulation correlation between transcription factors and enzyme genes in carotenoid transcription and metabolism Green graph indicates enzyme genes, purple graph indicates transcription factors, solid and dashed lines indicate positive and negative correlations, respectively; the thicker the line, the stronger the correlation

图9 RT-qPCR 分析不同生长时期金线莲叶片中的差异酶基因浅黄色柱状和S3代表3月龄，粉色柱状和S6代表6月龄，棕色柱状和S10代表10月龄。不同字母表示在0.05水平上差异性显著（P<0.05）

Fig. 9 RT-qPCR analysis of different enzymes genes in A. roxburghii leaves at different growth stages Light yellow bars and S3 indicate 3 months of age, pink bars and S6 indicate 6 months of age, and brown bars and S10 indicate 10 months of age. Different letters indicate significant differences at the 0.05 level（P < 0.05）

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