草莓花色苷物质鉴定及关键基因表达分析

doi:10.16420/j.issn.0513-353x.2022-0066

摘要/Abstract

摘要：

建立了应用HPLC-MS/MS快速分离、鉴定草莓花色苷的方法，并对不同果色12个草莓品种的成熟果实进行花色苷定量和定性分析，同时对比分析草莓（Fragaria × ananassa，八倍体）和森林草莓（F. vesca，二倍体）基因组中花色苷合成相关基因的数量和染色体定位，通过RNA-Seq和qRT-PCR分析它们在果实发育过程中的转录水平变化。在草莓中检测到8种花色苷，包括首次检测到的芍药素-3-葡萄糖苷、芍药素-丙二酰葡糖苷和芍药素-3-甲基丙二酰葡糖苷。不同果色草莓果实中总花色苷含量差异较大，均以天竺葵素-3-葡糖苷为主。草莓基因组包含73个花色苷合成相关基因，是森林草莓的3 ~ 4倍，均匀分布在4套亚基因组上。RNA-Seq结果显示整体上多拷贝基因在草莓果实中的表达水平没有显著差异，未发生明显的偏向性表达。花色苷合成关键路径基因（PAL1、CHS、CHI、F3H、DFR1、ANS、UFGT），转运基因（GST）和转录因子基因MYB10在草莓果实成熟过程中表达量显著增加，尤其是花色苷积累期，表明这些基因在草莓果实花色苷积累中起关键作用。

关键词: 草莓, 果实, 色泽, 花色苷, 多倍体

Abstract:

HPLC-MS/MS was applied to establish a method to isolate and identify anthocyanins in Fragaria × ananassa fruit，and anthocyanins from 12 strawberry cultivars with different fruit colors were analyzed qualitatively and quantitatively. Meanwhile，a comparative analysis was made on the number and chromosome location of genes related to anthocyanin synthesis between diploid(F. vesca)and octoploid(F. × ananassa)strawberry genome，and the transcription levels of which during fruit development were analyzed by RNA-Seq and qRT-PCR. In this study，totally eight anthocyanin components were detected，and peonidin-3-O-glucoside，peonidin-malonylglucoside and peonidin-3-methylmalonylglucoside were first detected in F. × ananassa. Pelargonidin-3-glucoside was the main anthocyanin in all 12 strawberry cultivars，while the total contents were different. Seventy-three genes related to anthocyanin synthesis were identified in the F. × ananassa genome distributed uniformly in four subgenomes，which were almost three-or four-fold than in the F. vesca genome. The results of RNA-Seq showed that on the whole there was no significant difference and biased expression between the expression levels of multi-copy genes in F.×ananassa fruit. The expression levels of key structural genes for anthocyanin synthesis(PAL1,CHS,CHI,F3H,DFR1,ANS,UFGT),transportation(GST)and transcription regulatory factor MYB10 were significantly increased during fruit development，especially during anthocyanin accumulation stages，suggesting that these genes play key roles in anthocyanin accumulation in strawberry fruits.

Key words: Fragaria × ananassa, fruit, color, anthocyanin, polyploid

中图分类号:

S668.1

袁华招, 庞夫花, 王静, 蔡伟建, 夏瑾, 赵密珍. 草莓花色苷物质鉴定及关键基因表达分析[J]. 园艺学报, 2023, 50(4): 791-801.

YUAN Huazhao, PANG Fuhua, WANG Jing, CAI Weijian, XIA Jin, ZHAO Mizhen. Identification of Anthocyanin Compositions and Expression Analysis of Key Related Genes in Fragaria × ananassa[J]. Acta Horticulturae Sinica, 2023, 50(4): 791-801.

图/表 8

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基因Gene	正向引物（5′-3′）Forword primer	反向引物（5′-3′）Reverse primer
FaPAL1	CAGTGACCAACCATGTCCAG	TCCTCCAAATGCCTCAAATC
FaCHS	GCCTGAGAAGTTAGAAGCCACG	CGAACCCAAACAGAACACCC
FaCHI	AGCATCACCCTCTACCCTCAT	ACCCACTGCACCCATAGCTG
FaF3H	TGTGGCGTTTGAGTCCGAGA	TGACGAGCTGATGGGGTTGG
FaDFR1	CAGGGTTTGAGTTCAAGTACAGC	TATCGCCATTCTCCTGCTTCT
FaANS	ATCTTCTCCTTGGGCGGCTC	AACATGGTTCCCGGTCTGCA
FaUFGT	CAAGCAATCCAACAGCTCAATC	GAAAACATACCCCTCCGGCAC
FaGST	CAAGTTCCAGCAATCGAAGA	TGGGAAGGATCACAAGTTGA
FaMYB10	GAGAAGGCAAATGGCATCAT	TTCATCCTCTGCAAACTCTCC

基因Gene	正向引物（5′-3′）Forword primer	反向引物（5′-3′）Reverse primer
FaPAL1	CAGTGACCAACCATGTCCAG	TCCTCCAAATGCCTCAAATC
FaCHS	GCCTGAGAAGTTAGAAGCCACG	CGAACCCAAACAGAACACCC
FaCHI	AGCATCACCCTCTACCCTCAT	ACCCACTGCACCCATAGCTG
FaF3H	TGTGGCGTTTGAGTCCGAGA	TGACGAGCTGATGGGGTTGG
FaDFR1	CAGGGTTTGAGTTCAAGTACAGC	TATCGCCATTCTCCTGCTTCT
FaANS	ATCTTCTCCTTGGGCGGCTC	AACATGGTTCCCGGTCTGCA
FaUFGT	CAAGCAATCCAACAGCTCAATC	GAAAACATACCCCTCCGGCAC
FaGST	CAAGTTCCAGCAATCGAAGA	TGGGAAGGATCACAAGTTGA
FaMYB10	GAGAAGGCAAATGGCATCAT	TTCATCCTCTGCAAACTCTCC

组分 Peak No.	保留时间/min Retention time	MS[M+H]/（m/z）	MS2/（m/z）	化合物 Compound
1	15.47	449	287	矢车菊素-3-葡糖苷^* Cyanidin-3-glucoside^*
2	16.86	433	271	天竺葵素-3-葡糖苷^* Pelargonidin-3-glucoside^*
3	17.73	463	301，286	芍药素-3-葡糖苷Peonidin-3-O-glucoside
4	19.12	535	287	矢车菊素-丙二酰葡糖苷Cyanidin- malonylglucoside
5	20.23	519	271	天竺葵素-丙二酰葡糖苷Pelargonidin-malonylglucoside
6	20.73	549	301，286	芍药素-丙二酰葡糖苷Peonidin-malonylglucoside
7	21.74	533	271	天竺葵素-3-甲基丙二酰基葡糖苷Pelargonidin-3-methylmalonylglucoside
8	22.05	563	301，286	芍药素-3-甲基丙二酰基葡糖苷Peonidin-3-methylmalonylglucoside

组分 Peak No.	保留时间/min Retention time	MS[M+H]/（m/z）	MS2/（m/z）	化合物 Compound
1	15.47	449	287	矢车菊素-3-葡糖苷^* Cyanidin-3-glucoside^*
2	16.86	433	271	天竺葵素-3-葡糖苷^* Pelargonidin-3-glucoside^*
3	17.73	463	301，286	芍药素-3-葡糖苷Peonidin-3-O-glucoside
4	19.12	535	287	矢车菊素-丙二酰葡糖苷Cyanidin- malonylglucoside
5	20.23	519	271	天竺葵素-丙二酰葡糖苷Pelargonidin-malonylglucoside
6	20.73	549	301，286	芍药素-丙二酰葡糖苷Peonidin-malonylglucoside
7	21.74	533	271	天竺葵素-3-甲基丙二酰基葡糖苷Pelargonidin-3-methylmalonylglucoside
8	22.05	563	301，286	芍药素-3-甲基丙二酰基葡糖苷Peonidin-3-methylmalonylglucoside

品种 Cultivar	各组分占比/% Percentage of each anthocyanin component
品种 Cultivar	1	2	3	4	5	6	7	8
四季香 Sijixiang	4.25 ± 0.18	81.11 ± 3.42	6.05 ± 0.26	ND	8.59 ± 0.36	ND	ND	ND
明旭Mingxu	3.32 ± 0.14	64.02 ± 2.71	6.15 ± 0.26	0.82 ± 0.03	22.93 ± 0.97	ND	2.76 ± 0.12	ND
97-17-1	5.47 ± 0.17	75.19 ± 2.28	7.18 ± 0.22	ND	12.16 ± 0.37	ND	ND	ND
97-4-17	5.11 ± 0.31	75.84 ± 4.54	8.52 ± 0.51	ND	10.53 ± 0.63	ND	ND	ND
甜查理 Sweet Charlie	3.24 ± 0.17	70.65 ± 3.66	10.07 ± 0.52	ND	12.35 ± 0.64	ND	3.69 ± 0.19	ND
幸香 Sachinoka	6.80 ± 0.49	79.23 ± 5.73	7.66 ± 0.55	ND	ND	0.87 ± 0.06	ND	5.44 ± 0.39
久59-1 Jiu 59-1	11.21 ± 0.69	78.46 ± 4.86	9.28 ± 0.57	ND	ND	1.05 ± 0.06	ND	ND
宁丰 Ningfeng	9.83 ± 0.43	75.53 ± 3.29	14.64 ± 0.64	ND	ND	ND	ND	ND
紫金久红 Zijin Jiuhong	9.97 ± 0.59	83.17 ± 4.96	ND	ND	ND	ND	ND	6.86 ± 0.41
香Xiang	10.54 ± 0.52	76.47 ± 3.75	12.99 ± 0.64	ND	ND	ND	ND	ND
枥木少女 Tochiotome	14.13 ± 0.92	72.38 ± 4.71	13.49 ± 0.88	1.23 ± 0.08	ND	ND	ND	ND
桃熏Tokun	14.00 ± 0.90	49.64 ± 3.20	15.27 ± 0.99	ND	21.09 ± 1.36	ND	ND	ND
平均值 Average	8.16 ± 0.46	73.47 ± 3.93	10.12 ± 0.50	1.03 ± 0.06	14.61 ± 0.72	0.96 ± 0.06	3.23 ± 0.15	6.15 ± 0.04