不同品种蝴蝶兰黄酮代谢途径分析

doi:10.3969/j.issn.1001-1803.2022.12.005

摘要/Abstract

摘要：

采用高效液相色谱（HPLC）研究18种蝴蝶兰的黄酮代谢途径。建立HPLC技术分析黄酮代谢途径（调控颜色）。使用荧光定量PCR（Real-Time PCR）进一步分析蝴蝶兰黄酮代谢途径关键基因表达水平；农杆菌介导瞬时转化蝴蝶兰，获得转基因株系。蝴蝶兰HPLC分离技术可成功分离29种组分，与其它物种葡萄、风信子差别较大；使用Real-Time PCR分析不同品种蝴蝶兰黄酮代谢途径关键基因，发现不同品种的查尔酮合酶（Chalcone synthase，CHS），黄烷酮-3-羟化酶（Flavonol-3-hydroxylase，F3H）以及查尔酮异构酶（Chalcone isomerase，CHI）时空表达模式基本一致；农杆菌介导法构建关键酶基因RNAi干扰载体瞬时转化蝴蝶兰，黄酮代谢途径中关键酶基因干扰表达会淡化蝴蝶兰颜色。建立了蝴蝶兰黄酮代谢途径的HPLC研究方法，并对其分子机制进行了初步研究，对蝴蝶兰花色调控机理的揭示提供了理论和实践基础。

关键词: 蝴蝶兰, 黄酮代谢, 高效液相色谱, 分子机制

Abstract:

High performance liquid chromatography （HPLC） was used to study the flavonoid metabolism pathways of 18 Phalaenopsis. A HPLC technology was established to analyze the pathway of flavonoid metabolism （regulate color）. Fluorescence quantitative PCR （Real-Time PCR） was used to further analyze the expression level of key genes in Phalaenopsis flavonoid synthesis pathway, and an Agrobacterium-mediated transient transformation of Phalaenopsis was used to obtain transgenic strains. Phalaenopsis HPLC separation technology can successfully separate 29 components, which is quite different from other species of grape and hyacinth. Real-Time PCR analysis of the key genes in the flavonoid synthesis pathway of different species of Phalaenopsis finds that the temporal and spatial expression patterns of Chalcone synthase （CHS）, Flavonol-3-hydroxylase （F3H） and Chalcone isomerase （CHI） of different species are basically the same. The key enzyme gene RNAi interference vector is constructed by Agrobacterium-mediated method to transform Phalaenopsis transiently. The interference expression of key enzyme genes in the flavonoid synthesis pathway will reduce the color of Phalaenopsis. In conclusion, the HPLC research method of Phalaenopsis flavonoid metabolism pathway is established, and its molecular mechanism is preliminary studied, which provids a theoretical and practical basis for revealing the color regulation mechanism of Phalaenopsis orchids.

Key words: Phalaenopsis, flavonoid metabolism, high performance liquid chromatography, molecular mechanism

中图分类号:

TQ658

程灵,汪建平. 不同品种蝴蝶兰黄酮代谢途径分析[J]. 日用化学工业（中英文）, 2022, 52(12): 1293-1299.

Cheng Ling,Wang Jianping. Analysis of flavonoid metabolic pathways of different varieties of Phalaenopsis and their molecular mechanism[J]. China Surfactant Detergent & Cosmetics, 2022, 52(12): 1293-1299.

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参考文献 13

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基因	序列	功能
PhF3'5'H-F	CGAACTCCAATGGCTTCTCC	Real-Time PCR
PhF3'5'H-R	CCCAACTCCTCTCAGCATACCT	Real-Time PCR
PhDFR-F	TTCAATCCAAAGACCCTGAGAAC	Real-Time PCR
PhDFR-R	TTACGCTGCCTGACCTCTTG	Real-Time PCR
PhF3'H-F	ATCGGACTTAACCGACTCGC	Real-Time PCR
PhF3'H-R	CAACACGTGGAAGAGAGAGC	Real-Time PCR
Tub-F	GCCTAAGGTCTCGGATACTGTT	Real-Time PCR
Tub-R	GCTCATGGTGGCAGATATGA	Real-Time PCR

组分	时间/min	分子结构	分子及碎片分子量
1	1.532 5	—	—
2	3.871	—	—
3	5.046	飞燕草素-3-O-芸香糖苷-7-O-葡萄糖苷	773/303
4	5.698	飞燕草素-3-O-葡萄糖苷	465/303
5	6.276	—	—
6	7.683	—	—
7	9.052	—	—
8	11.047	飞燕草素-3-O-咖啡酰葡萄糖苷-7-O-葡萄糖苷	789/627/465/303
9	12.032	—	—
10	13.684	—	—
11	15.673	天竺葵素-3-O-桑布糖苷	565/271
12	16.176	—	—
13	17.591	矢车菊素-3-O-丙二酰葡萄糖苷-7-O-葡萄糖苷-3’-O-葡萄糖苷	859/773/287
14	19.158	飞燕草素	303
15	20.464	矢车菊素-3-O-苹果酰葡萄糖苷-7-O-葡萄糖苷	—
16	21.389	矢车菊素-3-O-咖啡酰葡萄糖苷	611/449/287
17	22.648	矢车菊素-3-O-丙二酰槐糖苷-7-O-阿魏酰葡萄糖苷-3’-O-丙二酰葡萄糖苷	1283/1197/949/287
18	22.967	矢车菊素-3-O-咖啡酰阿拉伯糖苷	581
19	24.057	矢车菊素-3-O-槐糖苷-7-O-芥子酰葡萄糖苷-3’-O-对香豆酰葡萄糖苷	1287
20	25.685	天竺葵素-3-O-咖啡酰葡萄糖苷-7-O-对香豆酰葡萄糖苷	903/433/271
21	27.486	矢车菊素-3-O-葡萄糖苷-7-O-阿魏酰葡萄糖苷-3’-O-芥子酰葡萄糖苷	1155
22	30.562	天竺葵素-3-O-咖啡酰葡萄糖苷	595/433/271
23	34.018	矢车菊素-3-O-乙酰葡萄糖苷-7-O-芥子酰葡萄糖苷-3’-O-芥子酰葡萄糖苷	1227
24	31.281	—	—
25	31.893	矢车菊素-3-O-丙二酰葡萄糖苷-7-O-芥子酰葡萄糖苷-3’-O-芥子酰葡萄糖苷	1271
26	32.182	天竺葵素-3-O-葡萄糖苷-7-O-芥子酰葡萄糖苷	801/595/271
27	39.425	矢车菊素-3-O-丙二酰葡萄糖苷-7-O-阿魏酰葡萄糖苷	873/697/611
28	42.183	天竺葵素-3-O-对香豆酰葡萄糖苷-7-O-芥子酰葡萄糖苷	947/741/595/271
29	46.837	天竺葵素-3-O-丙二酰葡萄糖苷-7-O-芥子酰葡萄糖苷	887/681/433/271

品种	花青素含量/%			总花青素含量/%	总黄酮含量/%
品种	天竺葵素	矢车菊素	飞燕草素	总花青素含量/%	总黄酮含量/%
聚宝红玫瑰	34.57±0.11	60.23±1.14	3.63±0.18	0.82±0.09	3.15±0.08
红梅	39.39±0.35	59.52±1.22	0.57±0.04	1.15±0.07	2.68±0.12
花F1	33.64±0.28	65.34±1.37	0.65±0.05	0.71±0.04	1.42±0.06
翔凤	27.74±0.25	71.53±1.45	0.37±0.03	1.19±0.07	1.86±0.02
红彤	23.19±0.24	75.17±1.58	0.49±0.07	1.18±0.05	2.14±0.11
大富贵	29.31±0.28	70.46±1.78	0.00±0.00	0.48±0.02	1.27±0.05
金尚美人	34.40±0.39	62.16±1.27	1.87±0.23	0.86±0.05	2.15±0.07
红珍珠	21.08±0.14	76.36±0.45	1.12±0.24	0.82±0.09	2.28±0.06
新红龙	25.87±0.85	73.05±1.32	0.43±0.08	0.81±0.07	1.64±0.14
红钻石	28.37±1.34	69.62±2.57	0.74±0.05	1.06±0.10	2.02±0.06
S083	26.22±0.39	72.02±2.74	0.63±0.08	0.86±0.06	1.26±0.09
A4656	17.73±0.46	69.06±1.53	3.76±0.32	1.27±0.10	1.39±0.07
眉红	19.29±0.58	77.71±2.47	1.65±0.26	0.84±0.05	1.63±0.06
红天使	17.35±0.29	79.10±3.63	2.37±0.24	0.68±0.05	1.57±0.08
大辣椒	34.05±1.42	65.82±3.47	0.00±0.00	0.97±0.07	1.41±0.09
眉白	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	3.15±0.08
A5744	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	1.76±0.09
P48	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	1.07±0.04

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