不同温度LED光萎凋对铁观音MEP上游关键基因和香气的影响

doi:10.3864/j.issn.0578-1752.2020.02.009

摘要/Abstract

摘要：

【目的】萜类化合物是乌龙茶挥发性芳香物质的重要组分,2-C-甲基-D-赤藓醇-4-磷酸途径（MEP）上游关键基因直接参与调控萜类化合物前体物质的合成。而乌龙茶香气的形成与萎凋工序密切相关,光照和温度是影响萎凋的重要因子,探讨LED光与温度在乌龙茶萎凋过程中对香气的影响,为提高乌龙茶萎凋叶香气品质提供参考。【方法】基于转录组数据,根据KEGG筛选出响应光照的MEP上游关键基因（DXS、DXR、HDS、HDR）。对一芽三叶铁观音鲜叶进行LED白光和不同温度（20℃（L20）、25℃（L25）、30℃（L30）、35℃（L35）和40℃（L40））萎凋处理,黑暗下温度（20℃（D20）、25℃（D25）、30℃（D30）、35℃（D35）和40℃（D40））萎凋处理;分别测定铁观音萎凋叶的香气组分和MEP上游关键基因的相对表达量。【结果】L30处理萎凋叶各基因表达量达到最大值,萜类基因（DXS、DXR、HDS、HDR）表达量分别为XY组（对照）的4.31、5.28、11.77、1.59倍,为D30处理的2.24、2.39、1.86和1.60倍。D30组各基因表达量为黑暗处理组最大,依次为XY组的1.92、2.21、6.34和0.99倍。L20处理萎凋叶的α-法呢烯芳樟醇氧化物（I、II）含量最高,较XY依次提高了15.05%、4.92%和15.13%;L30处理萎凋叶的橙花叔醇、芳樟醇和香叶醇含量最高,较XY组依次提高了3.71%、6.14%和15.28%;LED组铁观音萎凋叶主要香气组分含量均高于相对应的温度处理组。通过主成分分析法建立数学模型,对萎凋叶香气组分进行评估,得出L20组萎凋叶得分最高,L30组萎凋叶次之;与香气分析得出结果一致。【结论】铁观音萎凋叶基因表达量与香气含量的变化趋势不存在同步性;L30处理萎凋叶基因表达量、主要萜类香气物质含量和主成分分析得分均较高,这与铁观音生产上的萎凋温度相一致。萎凋温度过高（40℃）不利于萎凋叶萜类关键基因的表达和萜类化合物的形成。

关键词: 铁观音, 萎凋, LED, 温度, 香气, DXS, DXR, HDS, HDR, 萜类化合物

Abstract:

【Objective】Terpenoids are important aroma components of oolong tea, and the key upstream genes of the MEP are directly involved in the regulation of the synthesis of terpenoid precursors. Withering, a process closely related to the aroma formation of oolong tea, is affected by many factors such as light exposure and temperature. The present study was aimed at investigating the effects of LED light and temperature on the aroma formation of oolong tea during withering process and to provide reference for improving aroma quality of withered leaves of Oolong tea. 【Method】 Firstly, the key upstream genes of MEP (DXS, DXR, HDS, and HDR) in tea which responded to light were selected according to our previous transcriptome data and KEGG pathways. The tea cultivar Tieguanyin was chosen as test materials, which were plucked in Tea Science Teaching and Research Base of Fujian Agriculture and Forestry University with one bud and three leaves. The freshly plucked leaves were treated under white LED light coupling with series temperature ((20℃ (L20), 25℃ (L25), 30℃ (L30), 35℃ (L35) and 40℃ (L40) ), Dark treatment (20℃ (D20), 25℃ (D25), 30℃ (D30), 35℃ (D35), and 40℃ (D40) ), and then the aroma contents and key upstream gene of MEP of withered leaves were determined.【Result】Under L30 treatment, DXS, DXR, HDS and HDR genes in the withered leaves were reached maximum, and which was 4.31, 5.28, 11.77, and 1.59 fold of XY (CK), respectively; these genes in L30 were 2.24, 2.39, 1.86 and 1.60 fold compared to D30 treatment; these genes expression were highest in D30 among Dark group and was 1.92, 2.21, 6.34 and 0.99 fold of XY, respectively. The contents of α-Farnesene and Linalool oxide (I, II) of L20 was the highest, which were increased by 15.05%, 4.92% and 15.13%, respectively, compared to XY. The highest content of Nerolidol, Linalool and Geraniol were occurred in L30 treatment, which were increased by 3.71%, 6.14% and 15.28%, respectively, compared with XY. The content of main aroma components in the LED group of Tieguanyin withered leaves was higher than that of temperature group. The mathematical model was established by principal component analysis method and the aroma components of the withered leaves were evaluated. It was found that the L20 treatment had the highest score and followed by L30, which were consistent with the aroma analysis. 【Conclusion】According to the comprehensive test results, there was no synchronization between the expression of the genes and the aroma contents of Tieguanyin, The gene expression under L30 treatment, aroma contents and principal component analysis scores were higher than that under other treatments. These results were consistent with the withering temperature in Tieguanyin production: when withering temperature was too high (40℃), the related gene expression and the formation of terpenoids in the withered leaves were prohibited.

Key words: Tieguanyin, withering leaves, LED, temperature, aroma, DXS, DXR, HDS, HDR, terpenoids

游芳宁,邓慧莉,胡娟,姚知灵,武帅强,秦艺嘉,汤铜华,孙云. 不同温度LED光萎凋对铁观音MEP上游关键基因和香气的影响[J]. 中国农业科学, 2020, 53(2): 346-356.

YOU FangNing,DENG HuiLi,HU Juan,YAO ZhiLing,WU ShuaiQiang,QIN YiJia,TANG TongHua,SUN Yun. Effects of LED Light Withering at Different Temperatures on Expression of Key Genes in the Upstream of MEP and Formation of Volatiles in Tieguanyin Tea[J]. Scientia Agricultura Sinica, 2020, 53(2): 346-356.

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https://www.chinaagrisci.com/CN/Y2020/V53/I2/346

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不同温度LED光照处理铁观音萎凋叶主要香气组分的相对含量"

香气组分 Volatile compounds	处理 Treatment						处理 Treatment
香气组分 Volatile compounds	XY	D20	D25	D30	D35	D40	XY	L20	L25	L30	L35	L40
芳醇氧化物II Linalool oxide II (X₁)	0.06	7.93	10.00	10.20	9.09	8.64	0.06	15.19	11.14	13.44	8.95	9.68
α-法呢烯 α-Farnesene (X₂)	0.08	7.15	4.83	6.24	6.69	3.54	0.08	15.13	5.14	8.56	6.99	4.18
香叶醇 Geraniol (X₃)	2.38	11.51	11.10	16.50	10.42	9.70	2.38	16.34	11.58	17.66	13.08	12.37
乙酸叶醇酯 3-Hexen-1-ol,acetate,(Z) (X₄)	2.32	12.58	8.15	14.44	5.79	4.76	2.32	15.18	10.28	13.57	12.51	10.41
芳樟醇 Linalool (X₅)	4.32	8.88	8.29	9.83	8.84	7.88	4.32	9.45	9.33	10.46	9.01	8.60
芳醇氧化物I Linalool oxide I (X₆)	2.33	4.32	5.33	4.51	4.96	4.91	2.33	7.25	6.99	6.43	5.63	5.39
顺式-己酸-3-己烯酯 Hexanoic acid,3-hexenyl ester,(Z) (X₇)	0.12	3.11	2.33	3.13	4.28	3.78	0.12	7.05	2.33	3.13	4.17	3.05
苯乙醇 Phenylethyl Alcohol (X₈)	0.48	0.92	2.56	2.24	1.45	2.29	0.48	3.57	2.60	3.15	1.62	1.87
橙花叔醇 Nerolidol (X₉)	0.33	2.53	2.93	3.56	3.36	2.40	0.33	3.54	3.40	4.04	3.51	2.96
脱氢芳樟醇 1,5,7-Octatrien-3-ol,3,7-dimethyl (X₁₀)	0.95	3.39	2.89	2.79	3.69	2.29	0.95	3.49	3.92	4.11	3.82	1.40
吲哚 Indole (X₁₁)	1.20	1.76	2.37	2.13	3.23	2.36	1.20	3.12	3.02	3.39	3.50	2.59
叶醇 3-Hexen-1-ol (X₁₂)	0.87	1.09	1.01	0.80	2.17	2.01	0.87	2.99	2.05	2.04	3.97	3.47
水杨酸甲酯 Methyl salicylate (X₁₃)	0.14	1.86	1.99	2.54	2.66	2.81	0.14	2.36	2.81	3.01	3.42	3.83
丁酸-3-己烯酯 Butanoic acid,3-hexenyl ester,(E) (X₁₄)	0.05	0.80	1.40	0.91	2.38	2.22	0.05	1.69	1.15	1.66	2.12	2.35
香叶基丙酮 5,9-Undecadien-2-one,6,10-dimethyl-,(E) (X₁₅)	0.66	1.20	0.96	0.77	0.67	1.18	0.66	1.33	0.99	0.81	0.70	0.68
2-庚醇 2-Heptanol (X₁₆)	0.08	1.31	2.63	1.45	1.40	1.87	0.08	1.08	2.12	1.23	1.29	1.24
己酸己酯 Hexanoic acid,hexyl ester (X₁₇)	0.15	0.36	0.30	0.51	0.39	0.25	0.15	0.92	0.21	0.26	0.31	0.15
顺-3-己烯酸顺-3-己烯酯 cis-3-Hexenyl, cis-3-hexenoate (X₁₈)	0.19	0.45	0.35	0.37	0.44	0.44	0.19	0.85	0.30	0.36	0.47	0.41
2-甲基丁酸顺-3-己烯酯cis-3-Hexenyl-2-methylbutyrate (X₁₉)	0.28	0.47	0.23	0.33	0.36	0.18	0.28	0.64	0.24	0.30	0.35	0.15
苯甲醇 Benzyl alcohol (X₂₀)	1.33	1.00	0.52	0.71	0.55	0.53	1.33	0.63	0.67	0.47	0.54	0.61
1,2-苯二甲酸双（2-甲基丙基）酯 1,2-Benzenedicarboxylic acid, bis (2-methylpropyl) ester (X₂₁)	0.87	0.29	0.45	0.39	0.29	0.43	0.87	0.58	0.26	0.27	0.26	0.16
十四烷 Tetradecane (X₂₂)	0.17	0.38	0.35	0.29	0.24	0.30	0.17	0.47	0.24	0.19	0.20	0.31
反式2-己烯基己酸 Hexanoic acid,2-hexenyl ester,(E) (X₂₃)	0.17	0.17	0.15	0.26	0.22	0.15	0.17	0.43	0.11	0.15	0.17	0.10
2-硝基乙基苯 Benzene,(2-nitroethyl) (X₂₄)	0.04	0.31	0.35	0.42	0.47	0.27	0.04	0.43	0.38	0.47	0.44	0.34
2,2,3,3-四甲基丁烷 2,2,3,3-tetramethyl butane (X₂₅)	0.28	0.42	0.23	0.38	0.19	0.36	0.28	0.22	0.05	0.01	0.17	0.02

表3

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表4

表5

表6

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处理名称 Treatment name	样品处理方法 Sample processing method
XY	铁观音鲜叶 Fresh leaves
L20	LED白光+20℃条件下萎凋60 min Withered 60 min at condition of LED white light and 20℃
L25	LED白光+25℃条件下萎凋60 min Withered 60 min at condition of LED white light and 25℃
L30	LED白光+30℃条件下萎凋60 min Withered 60 min at condition of LED white light and 30℃
L35	LED白光+35℃条件下萎凋60 min withered 60 min at condition of LED white light and 35℃
L40	LED白光+40℃条件下萎凋60 min Withered 60 min at condition of LED white light and 40℃
D20	20℃无光条件下萎凋60 min Withered 60 min at condition of dark and 20℃
D25	25℃无光条件下萎凋60 min Withered 60 min at condition of dark and 25℃
D30	30℃无光条件下萎凋60 min Withered 60 min at condition of dark and 30℃
D35	35℃无光条件下萎凋60 min withered 60min at condition of dark and 35℃
D40	40℃无光条件下萎凋60 min withered 60 min at condition of dark and 40℃

引物名称 Primer name	序列 Sequence (5'→3')	用途 Function
DXS-qF	GGATGGTGGGTGGTTCAG	实时荧光定量PCR qRT-PCR
DXS-qR	ATGAGAACAGGTCCAGGTGC	实时荧光定量PCR qRT-PCR
DXR-qF	CGGGATAAACTACCTTGACATT	实时荧光定量PCR qRT-PCR
DXR-qR	TTCCTCGCCCACAAGTCAT	实时荧光定量PCR qRT-PCR
HDS-qF	AACCCTCTCTTTCTCTCTCTCG	实时荧光定量PCR qRT-PCR
HDS-qR	TCTGTTGTGGTCATCGTTTG	实时荧光定量PCR qRT-PCR
HDR-qF	GTGTTGAGAGGGCAGTCCA	实时荧光定量PCR qRT-PCR
HDR-qR	AGCAGGCAGAACCACGACA	实时荧光定量PCR qRT-PCR
GDPDH-qF	TTTGGTGAGAAAGCAGTAG	内参基因Reference gene
GDPDH-qR	TGGGCAGCAGCCTTATCCT	内参基因Reference gene

主成分 Principal component	特征值 Eigenvalue	方差贡献率 Variance contribution rate (%)	累计贡献率 Cumulative contribution rate (%)
F₁	12.842	51.367	51.367
F₂	5.345	21.379	72.746
F₃	1.992	7.968	80.714
F₄	1.646	6.586	87.300
F₅	1.102	4.408	91.708

主成分 Principal component	X₁	X₂	X₃	X₄	X₅	X₆	X₇	X₈	X₉	X₁₀	X₁₁	X₁₂	X₁₃	X₁₄	X₁₅	X₁₆	X₁₇	X₁₈	X₁₉	X₂₀	X₂₁	X₂₂	X₂₃	X₂₄	X₂₅
F₁	0.96	0.85	0.89	0.76	0.90	0.89	0.84	0.78	0.93	0.73	0.83	0.54	0.73	0.61	0.35	0.41	0.60	0.72	0.41	-0.80	-0.58	0.43	0.42	0.91	-0.34
F₂	0.01	0.47	0.00	0.18	-0.17	-0.12	0.37	0.05	-0.25	-0.06	-0.37	-0.29	-0.57	-0.44	0.60	-0.34	0.76	0.57	0.81	0.44	0.63	0.63	0.82	-0.21	0.62
F₃	0.16	-0.11	0.17	0.13	0.26	0.05	-0.28	0.21	0.13	0.20	-0.29	-0.69	-0.13	-0.36	0.38	0.67	-0.08	-0.29	-0.18	-0.07	-0.13	0.23	-0.22	0.04	0.32
F₄	0.02	-0.13	-0.26	-0.41	-0.22	0.07	0.22	0.12	-0.19	-0.36	-0.07	0.20	0.16	0.47	0.42	0.40	-0.03	0.26	-0.28	-0.27	0.03	0.45	-0.07	-0.21	0.14
F₅	-0.16	-0.05	0.07	0.15	0.16	-0.35	0.14	-0.51	0.07	-0.01	-0.16	0.04	0.23	0.19	-0.09	-0.03	-0.01	0.07	0.09	0.04	-0.42	0.14	-0.07	0.15	0.51

处理 Treatment	F₁	F₂	F₃	F₄	F₅	F	排序Ranking
L20	92.18	8.57	5.16	-11.13	-0.98	48.85	1
L30	81.68	1.06	7.69	-11.91	-0.33	42.02	2
D30	69.21	2.70	8.30	-11.75	1.42	36.10	3
L35	69.82	0.06	3.48	-8.99	1.28	35.64	4
L25	65.62	-0.91	6.98	-8.16	-1.06	33.51	5
L40	61.44	-1.91	3.70	-6.54	0.87	31.07	6
D20	58.48	4.04	6.32	-9.82	1.57	30.85	7
D35	59.71	-0.07	3.41	-5.74	0.22	30.58	8
D25	57.31	0.17	7.13	-6.42	-0.88	29.60	9
D40	51.69	-0.94	4.10	-3.31	-0.37	26.46	10
对照 XY	12.17	0.94	1.34	-2.51	-0.13	6.39	11