外源褪黑素影响萝卜生长及对链格孢菌抗性的机理研究

doi:10.16420/j.issn.0513-353x.2021-0423

摘要/Abstract

摘要：

以‘江南圆白’萝卜（Raphanus sativus）幼苗为材料,叶片喷施等量0、50、100、500、1 000和1 500 μmol · L^-1褪黑素,测定各处理幼苗生物量、总叶绿素含量、APX酶活性、H₂O₂含量,内源褪黑素含量以及亚细胞结构,同时对各处理幼苗接种芸薹链格孢菌（Alternaria）后的发病情况进行统计。结果表明,在50、500 μmol · L^-1褪黑素浓度范围内,幼苗生长和抗性随喷施浓度升高而增加,其中500 μmol · L^-1处理各项指标最佳,而1 000、1 500 μmol · L^-1褪黑素处理降低各项指标,说明褪黑素可通过剂量依赖模式调节萝卜生长及抗性。此外对0、500和1 500 μmol · L^-1褪黑素处理的样品进行RNA-Seq转录组分析,筛选出8个与光合调控（Ubr）和逆境抗性（Aos1、Prp1、Zat-like、Jub1-like、Edr1、Usp和Eda）相关的差异表达基因,且其表达量亦呈明显的剂量依赖效应。

关键词: 萝卜, 褪黑素, 链格孢菌, 抗性, 转录组

Abstract:

In this study,the seedlings leaves of‘Jiangnan Yuanbai’radish（Raphanus sativus）were sprayed with 0,50,100,1 000 and 1 500 μmol · L^-1 melatonin solution,the effects of exogenous melatonin on plant biomass,total chlorophyll content,APX enzyme activity,content of H₂O₂,endogenous melatonin and subcellular structures were measured,disease indexes after Alternaria brassicae inoculation were also detected. It was found that within the concentrations of 50,500 μmol · L^-1 melatonin increased growth and resistence of seedling with the increase of concentration,and 500 μmol · L^-1 treatment showed the best performance,while the indexes of 1 000 μmol · L^-1 and 1 500 μmol · L^-1 treatments decreased sharply. The results showed that the melatonin influences radish growth and resistence relying on a dose-dependent effect.RNA-Seq based transcriptome analysis was conducted on 0,500 and 1 500 μmol · L^-1melatonin treated samples,eight key DEGs were selected,which were Ubr,acting on photosynthetic regulation,and Aos1,Prp1,Zat-like,Jub1-like,Edr1,Usp and Eda,regulating resistence. The expression pattern of DEGs was consistent with the phenotypic changes of all treatments,showed a dose-dependent pattern.

Key words: Raphanus sativus, melatonin, Alternaria brassicae, resistence, transcriptome

中图分类号:

S631.1

夏铭, 李经纬, 罗章瑞, 祖贵东, 王娅, 张万萍. 外源褪黑素影响萝卜生长及对链格孢菌抗性的机理研究[J]. 园艺学报, 2022, 49(3): 548-560.

XIA Ming, LI Jingwei, LUO Zhangrui, ZU Guidong, WANG Ya, ZHANG Wanping. Research on Mechanism of Exogenous Melatonin Effects on Radish Growth and Resistence to Alternaria brassicae[J]. Acta Horticulturae Sinica, 2022, 49(3): 548-560.

图/表 9

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基因名 Gene ID	基因名 Gene name	描述 Description	KEGG通路ID KEGG pathway ID	引物序列（5′-3′） Primer sequence
108828933	Aos1	丙二烯氧化物合酶,叶绿体 Allene oxide synthase,chloroplastic	Ko00592：α-亚麻酸代谢 α-Linolenic acid metabolism	F：ACCTACATGCCCTCGACGGATC R：TTGGCGTGTTTCGGCTCTGATG
108859130	Prp1	致病相关类蛋白1 Pathogenesis-related protein 1-like	Ko04626：植物—病菌互作 Plant-pathogen interaction	F：AACCAAGCACGACAGGCAGTAAG R：ACTCTTAGTCGGTCGGCGTAGC
108818819	At-like	锌指类蛋白ZAT10 Zinc finger protein ZAT10-like	无Naught	F：TCCACCACCAGAAGAACCTCACC R：GCACTTGTAGGACGACTTCTCAGC
108834050	Jub1-like	JUNGBRUNNEN类转录因子1 Transcription factor JUNGBRUNNEN 1-like	Ko04141：内质网中的蛋白质加工Protein processing in endoplasmic reticulum	F：CACAAGCCACACGAGCCACAG R：AATCTCCACCGTCGAGGTTCCC
108862765	Edr1	丝氨酸/苏氨酸蛋白激酶EDR1 Serine/threonine-protein kinase EDR1	无Naught	F：TTCGCAGACCTTAACCCGTTTCAG R：TGCAGGAGACTTACCAACCAGAGG
108848467	Ubr	紫外线B受体UVR8亚型X1 Ultraviolet-B receptor UVR8 isoform X1	Ko04120：泛素介导的蛋白质水解 Ubiquitin mediated proteolysis	F：AGTCACCGTTCTCCTCCTTCTCG R：TTGTTGCGTGGACTGCTCGATTAG
108860024	Usp	普遍应激蛋白A样蛋白 Universal stress protein A-like protein	无Naught	F：ACGAAAGCGAAGAGAGCATGGAAG R：CCCTGCGGTGTCAATGGAAGAG
108807199	Ech-like	类内源几丁质酶At2g43590 Endochitinase At2g43590-like	Ko00520：氨基糖和核苷酸糖代谢 Amino sugar and nucleotide sugar metabolism	F：CCTCGGGCGTATGTTGTAGTCAG R：GCAAGGACCTGATCGGCAACC
108847559	Actin-1 Actin-2	actin-7-like actin-7-like	无Naught 无Naught	F：GCATCACACTTTCTACAAC R：CCTGGATAGCAACATACAT F：GCAAGAGCTGGATACCGCAAAG R：CGATGAGCGATGGCTGGAAC

基因名 Gene ID	基因名 Gene name	描述 Description	KEGG通路ID KEGG pathway ID	引物序列（5′-3′） Primer sequence
108828933	Aos1	丙二烯氧化物合酶,叶绿体 Allene oxide synthase,chloroplastic	Ko00592：α-亚麻酸代谢 α-Linolenic acid metabolism	F：ACCTACATGCCCTCGACGGATC R：TTGGCGTGTTTCGGCTCTGATG
108859130	Prp1	致病相关类蛋白1 Pathogenesis-related protein 1-like	Ko04626：植物—病菌互作 Plant-pathogen interaction	F：AACCAAGCACGACAGGCAGTAAG R：ACTCTTAGTCGGTCGGCGTAGC
108818819	At-like	锌指类蛋白ZAT10 Zinc finger protein ZAT10-like	无Naught	F：TCCACCACCAGAAGAACCTCACC R：GCACTTGTAGGACGACTTCTCAGC
108834050	Jub1-like	JUNGBRUNNEN类转录因子1 Transcription factor JUNGBRUNNEN 1-like	Ko04141：内质网中的蛋白质加工Protein processing in endoplasmic reticulum	F：CACAAGCCACACGAGCCACAG R：AATCTCCACCGTCGAGGTTCCC
108862765	Edr1	丝氨酸/苏氨酸蛋白激酶EDR1 Serine/threonine-protein kinase EDR1	无Naught	F：TTCGCAGACCTTAACCCGTTTCAG R：TGCAGGAGACTTACCAACCAGAGG
108848467	Ubr	紫外线B受体UVR8亚型X1 Ultraviolet-B receptor UVR8 isoform X1	Ko04120：泛素介导的蛋白质水解 Ubiquitin mediated proteolysis	F：AGTCACCGTTCTCCTCCTTCTCG R：TTGTTGCGTGGACTGCTCGATTAG
108860024	Usp	普遍应激蛋白A样蛋白 Universal stress protein A-like protein	无Naught	F：ACGAAAGCGAAGAGAGCATGGAAG R：CCCTGCGGTGTCAATGGAAGAG
108807199	Ech-like	类内源几丁质酶At2g43590 Endochitinase At2g43590-like	Ko00520：氨基糖和核苷酸糖代谢 Amino sugar and nucleotide sugar metabolism	F：CCTCGGGCGTATGTTGTAGTCAG R：GCAAGGACCTGATCGGCAACC
108847559	Actin-1 Actin-2	actin-7-like actin-7-like	无Naught 无Naught	F：GCATCACACTTTCTACAAC R：CCTGGATAGCAACATACAT F：GCAAGAGCTGGATACCGCAAAG R：CGATGAGCGATGGCTGGAAC

褪黑素/（μmol · L^-1） Melatonin	株高/cm Plant height	茎粗/mm Stem diameter	鲜质量/g Fresh weight	干质量/g Dry weight
ddH₂O（对照Control）	13.57 ± 0.19 d	3.37 ± 0.04 cd	126.33 ± 0.93 d	10.75 ± 0.05 d
50	14.47 ± 0.18 c	3.73 ± 0.03 b	130.07 ± 0.87 c	11.81 ± 0.08 c
100	15.33 ± 0.22 b	3.90 ± 0.03 ab	139.17 ± 0.83 b	12.34 ± 0.06 b
500	16.11 ± 0.10 a	4.05 ± 0.07 a	156.27 ± 0.83 a	12.76 ± 0.04 a
1 000	14.79 ± 0.14 c	3.52 ± 0.05 c	126.53 ± 0.45 d	10.78 ± 0.16 d
1 500	12.91 ± 0.08 e	3.29 ± 0.03 d	123.61 ± 0.66 e	10.41 ± 0.10 e

褪黑素/（μmol · L^-1） Melatonin	株高/cm Plant height	茎粗/mm Stem diameter	鲜质量/g Fresh weight	干质量/g Dry weight
ddH₂O（对照Control）	13.57 ± 0.19 d	3.37 ± 0.04 cd	126.33 ± 0.93 d	10.75 ± 0.05 d
50	14.47 ± 0.18 c	3.73 ± 0.03 b	130.07 ± 0.87 c	11.81 ± 0.08 c
100	15.33 ± 0.22 b	3.90 ± 0.03 ab	139.17 ± 0.83 b	12.34 ± 0.06 b
500	16.11 ± 0.10 a	4.05 ± 0.07 a	156.27 ± 0.83 a	12.76 ± 0.04 a
1 000	14.79 ± 0.14 c	3.52 ± 0.05 c	126.53 ± 0.45 d	10.78 ± 0.16 d
1 500	12.91 ± 0.08 e	3.29 ± 0.03 d	123.61 ± 0.66 e	10.41 ± 0.10 e

褪黑素/（μmol · L^-1） Melatonin	叶片褪黑素含量/（ng · mL^-1） Melatonin content of leaf	SPAD	H₂O₂含量/（mmol · g^-1 FW） H₂O₂ content	APX活性/（μmol · g^-1 FW） APX activity
ddH₂O（对照Control）	14.36 ± 1.12 c	32.66 ± 0.54 d	7.35 ± 0.12 a	0.372 ± 0.009 e
50	—	34.98 ± 0.24 c	6.37 ± 0.08 b	0.551 ± 0.037 d
100	—	36.94 ± 0.23 b	5.64 ± 0.08 c	0.752 ± 0.017 c
500	19.23 ± 0.60 b	38.49 ± 0.27 a	4.69 ± 0.17 d	1.070 ± 0.023 a
1 000	—	35.58 ± 0.06 c	5.73 ± 0.06 c	0.863 ± 0.034 b
1 500	25.04 ± 0.82 a	32.79 ± 0.63 d	6.51 ± 0.14 b	0.422 ± 0.005 e