龙眼褪黑素合成途径SNAT、ASMT和COMT家族基因鉴定及表达分析

doi:10.16420/j.issn.0513-353x.2021-0003

园艺学报 ›› 2022, Vol. 49 ›› Issue (5): 1031-1046.doi: 10.16420/j.issn.0513-353x.2021-0003

龙眼褪黑素合成途径SNAT、ASMT和COMT家族基因鉴定及表达分析

肖学宸¹, 刘梦雨¹, 蒋梦琦¹, 陈燕¹, 薛晓东¹, 周承哲¹, 吴兴健¹, 吴君楠¹, 郭寅生¹, 叶开温², 赖钟雄¹, 林玉玲¹^,^*()

¹福建农林大学园艺植物生物工程研究所,福州 350002
²台湾大学植物科学研究所,中国台北 106

收稿日期:2021-08-01 修回日期:2021-09-07 出版日期:2022-05-25 发布日期:2022-05-25
通讯作者: 林玉玲 E-mail:buliang84@163.com
基金资助:
国家自然科学基金项目(31672127);福建省自然科学基金项目(2020J01543);福建省高原学科建设经费项(102/71201801101);福建农林大学创新基金项目(CXZX2019033S);福建农林大学创新基金项目(CXZX2018078);福建农林大学乡村振兴服务团队项目(11899170112)

Whole-genome Identification and Expression Analysis of SNAT,ASMT and COMT Families of Melatonin Synthesis Pathway in Dimocarpus longan

XIAO Xuechen¹, LIU Mengyu¹, JIANG Mengqi¹, CHEN Yan¹, XUE Xiaodong¹, ZHOU Chengzhe¹, WU Xingjian¹, WU Junnan¹, GUO Yinsheng¹, YEH Kaiwen², LAI Zhongxiong¹, LIN Yuling¹^,^*()

¹Institute of Horticultural Biotechnology,Fujian Agriculture and Forestry University,Fuzhou 350002,China
²Institute of Plant Science,Taiwan University,Taipei 106,China

Received:2021-08-01 Revised:2021-09-07 Online:2022-05-25 Published:2022-05-25
Contact: LIN Yuling E-mail:buliang84@163.com

摘要/Abstract

摘要：

基于第3代龙眼（Dimocarpus longan）基因组及转录组数据库,对褪黑素合成路径上可能的限速基因SNAT（serotonin N-acetyltransferase）、ASMT（N-acetylserotonin methyltransferase）和COMT（caffeic acid O-methyltransferase）进行基因家族成员鉴定,利用实时荧光定量PCR技术研究其在龙眼体胚早期的表达谱,并研究外源IAA、GA₃、MeJA、SA和ABA对龙眼胚性愈伤组织中褪黑素含量的影响,以及褪黑素合成途径相关基因的表达模式。结果显示：龙眼SNAT（DlSNAT）、ASMT（DlASMT）和COMT（DlCOMT）基因家族分别含有2、18和7个成员,蛋白质结构域保守,相同家族的成员之间保守基序相差极小,DlASMT和DlCOMT家族成员高度相似。基于氨基酸序列进行系统进化树分析,龙眼、拟南芥、水稻、小麦、番茄、辣椒和卷柏的SNAT家族可分为3个亚组;ASMT和COMT家族具有高度同源性,共同建树并分为3个亚组。DlSNAT、DlASMT和DlCOMT家族成员的启动子中含有大量响应生长素、赤霉素、茉莉酸甲酯、水杨酸和脱落酸的顺式作用元件。表达谱分析显示,DlASMT和DlCOMT家族成员主要在龙眼不完全胚性紧实结构和球形胚阶段高表达,DlSNAT1和DlSNAT2在龙眼体胚发生早期始终处于高水平表达。用0.1 mmol · L^-1IAA处理龙眼胚性愈伤组织,内源褪黑素含量在24 h内显著降低,GA₃、MeJA、SA和ABA处理则显著提高了褪黑素含量;同时分析DlSNAT、DlASMT、DlCOMT成员表达情况,内源褪黑素含量变化趋势和DlSNAT表达基本一致。综上,DlSNAT、DlASMT和DlCOMT可能通过影响内源褪黑素的合成参与调控龙眼早期体胚发生。

关键词: 龙眼, 褪黑素, SNAT, ASMT, COMT, 全基因组鉴定, 表达分析

Abstract:

In this study,on the basis of the third-generation longan（Dimocarpus longan Lour.）genome and transcriptome database,the possible rate-limiting genes of melatonin synthesis：SNAT（serotonin N-acetyltransferase）,ASMT（N-acetylserotonin methyltransferase）and COMT（caffeic acid O-methyltransferase）family genes were identified and analyzed. The effect of different exogenous phytohormones on the melatonin content was detected,and,the expression patterns of SNAT,ASMT,and COMT genes in respond to exogenous phytohormone treaments and during early somatic embryogenesis（SE）in longan were determined by quantitative real time polymerase chain reaction（qRT-PCR）. The results showed that the longan SNAT（DlSNAT）,ASMT（DlASMT）,and COMT（DlCOMT）family genes have two,18,and seven members,respectively;the protein domains and motifs in each same gene family are conserved,and DlASMTs and DlCOMTs are highly similar;phylogenetic analysis showed that the SNAT family of Dimocarpus longan,Arabidopsis thaliana,Oryza sativa,Triticum aestivum,Solanum lycopersicum,Capsicum annuum,and Selaginella tamariscina could be divided into three subgroups,while the ASMT and COMT families were highly homology and were divided into three subgroups;the cis-acting elements analysis revealed that a large number of auxin-,gibberellin-,methyl jasmonate-,salicylic acid-,and abscisic acid-related elements existed in the promoters of DlSNAT,DlASMT and DlCOMT family members;qRT-PCR analysis showed that most of DlASMTs and DlCOMTs were highly expressed in the incomplete embryonic compact structure and globular embryo stages of somatic embryo（SE）,while DlSNAT1 and DlSNAT2 were highly expressed during early SE in longan;the content of endogenous melatonin was significantly reduced under exogenous 0.1 mmol · L^-1of indole-3-acetic acid（IAA）treatment,whereas significantly increased under exogenous gibberellin A3（GA₃）,methyl jasmonate（MeJA）,salicylicacid（SA）and abscisic acid（ABA）treatments;at the same time,the expression changes of DlSNATs consist with the change trends of endogenous melatonin content. The results indicated that DlSNATs,DlASMTs and DlCOMTs may involved in the synthesis of endogenous melatonin thereby regulate the process of early SE in longan.

Key words: longan, melatonin, SNAT, ASMT, COMT, whole-genome identification, expression analysis

中图分类号:

S667.2

肖学宸, 刘梦雨, 蒋梦琦, 陈燕, 薛晓东, 周承哲, 吴兴健, 吴君楠, 郭寅生, 叶开温, 赖钟雄, 林玉玲. 龙眼褪黑素合成途径SNAT、ASMT和COMT家族基因鉴定及表达分析[J]. 园艺学报, 2022, 49(5): 1031-1046.

XIAO Xuechen, LIU Mengyu, JIANG Mengqi, CHEN Yan, XUE Xiaodong, ZHOU Chengzhe, WU Xingjian, WU Junnan, GUO Yinsheng, YEH Kaiwen, LAI Zhongxiong, LIN Yuling. Whole-genome Identification and Expression Analysis of SNAT,ASMT and COMT Families of Melatonin Synthesis Pathway in Dimocarpus longan[J]. Acta Horticulturae Sinica, 2022, 49(5): 1031-1046.

图/表 11

参考文献 35

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引物名称 Primer name	引物序列 Primer sequence	引物名称 Primer name	引物序列 Primer sequence
DlSNAT1-F	TTCATTCTCCACCGCAC	DlASMT16-F	CCCTCTGATGCCTCTCTAAG
DlSNAT1-R	CTCCATCCAAAGCAGACTG	DlASMT16-R	TCACGAACGGCTCTGAT
DlSNAT2-F	GCCACATTGCATTCTGTTAG	DlASMT12-F	CAAGCAATGACCAGTGACTC
DlSNAT2-R	GACCTTGACCCTGATAGCTAG	DlASMT12-R	CTTCCTCCTACATCAACCAATG
DlASMT6-F	TTGTTGGTTGCTCTGTGG	DlCOMT6-F	CGCCTTTGATTCTGATACAG
DlASMT6-R	CTCCTTCAAGAACGGCAT	DlCOMT6-R	GCCATCGTTGAACATCTTG
DlASMT4-F	CGAACGCATTTGAGTATCC	DlCOMT1-F	TCGGATGGGAAAGTTGAG
DlASMT4-R	TGTGAGGGTATTTGGCAGT	DlCOMT1-R	TGCCTTGTTGAATGGGA
DlASMT2-F	GTTCAACCACACAACCATAGTC	DlCOMT7-F	CCATTTGTGCGTCTCATTC
DlASMT2-R	CCAACATCCACAATCTGCT	DlCOMT7-R	AGTCAGAGTCAGTGTTCATTGC
DlASMT11-F	CAGGGCTTATGAGACAACACT	DlCOMT3-F	TTACCCTTTCAGAGTTGGCT
DlASMT11-R	CCACATCAACCAGGGAAT	DlCOMT3-R	AGAAGTTGGTGGTGGCAT

引物名称 Primer name	引物序列 Primer sequence	引物名称 Primer name	引物序列 Primer sequence
DlSNAT1-F	TTCATTCTCCACCGCAC	DlASMT16-F	CCCTCTGATGCCTCTCTAAG
DlSNAT1-R	CTCCATCCAAAGCAGACTG	DlASMT16-R	TCACGAACGGCTCTGAT
DlSNAT2-F	GCCACATTGCATTCTGTTAG	DlASMT12-F	CAAGCAATGACCAGTGACTC
DlSNAT2-R	GACCTTGACCCTGATAGCTAG	DlASMT12-R	CTTCCTCCTACATCAACCAATG
DlASMT6-F	TTGTTGGTTGCTCTGTGG	DlCOMT6-F	CGCCTTTGATTCTGATACAG
DlASMT6-R	CTCCTTCAAGAACGGCAT	DlCOMT6-R	GCCATCGTTGAACATCTTG
DlASMT4-F	CGAACGCATTTGAGTATCC	DlCOMT1-F	TCGGATGGGAAAGTTGAG
DlASMT4-R	TGTGAGGGTATTTGGCAGT	DlCOMT1-R	TGCCTTGTTGAATGGGA
DlASMT2-F	GTTCAACCACACAACCATAGTC	DlCOMT7-F	CCATTTGTGCGTCTCATTC
DlASMT2-R	CCAACATCCACAATCTGCT	DlCOMT7-R	AGTCAGAGTCAGTGTTCATTGC
DlASMT11-F	CAGGGCTTATGAGACAACACT	DlCOMT3-F	TTACCCTTTCAGAGTTGGCT
DlASMT11-R	CCACATCAACCAGGGAAT	DlCOMT3-R	AGAAGTTGGTGGTGGCAT

基因ID Gene-ID	基因名称 Gene name	染色体定位 Chromosome location	氨基酸数 Amino acid number	分子量/kD Molecular weight	理论等电点 Theoretical pI	不稳定系数 The Instabi- lity index	亲水性 GRAVY	亚细胞定位 Subcellular location	信号肽 Signal peptide
Dlo018311	DlSNAT1	8	187	20.81	9.73	33.60	0.064	过氧化物酶体Peroxisome	无No
Dlo030488	DlSNAT2	14	225	24.68	4.89	44.33	-0.070	叶绿体Chloroplast	无No
Dlo004250	DlASMT1	2	347	38.76	5.22	39.04	0.077	细胞质Cytoplasm	无No
Dlo011828	DlASMT2	5	389	43.13	6.02	35.12	0.000	细胞质Cytoplasm	无No
Dlo011829	DlASMT3	5	442	49.48	5.77	41.16	-0.223	细胞质Cytoplasm	无No
基因ID Gene-ID	基因名称 Gene name	染色体定位 Chromosome location	氨基酸数 Amino acid number	分子量/kD Molecular weight	理论等电点 Theoretical pI	不稳定系数 The Instabi- Lity index	亲水性 GRAVY	亚细胞定位 Subcellular location	信号肽 Signal peptide
Dlo011833	DlASMT4	5	365	40.35	5.39	33.80	-0.099	细胞质Cytoplasm	无No
Dlo011834	DlASMT5	5	355	38.89	5.55	32.36	0.090	细胞质Cytoplasm	无No
Dlo011835	DlASMT6	5	354	39.07	5.55	35.08	0.007	细胞质Cytoplasm	无No
Dlo017622	DlASMT7	8	244	27.16	5.59	29.58	-0.029	细胞骨架Cytoskeleton	无No
Dlo017625	DlASMT8	8	367	41.17	5.20	36.62	-0.096	细胞骨架Cytoskeleton	无No
Dlo017687	DlASMT9	8	365	40.99	5.45	32.12	-0.074	细胞质Cytoplasm	无No
Dlo017690	DlASMT10	8	364	40.91	5.52	31.91	-0.074	叶绿体Chloroplast	无No
Dlo020696	DlASMT11	9	370	41.10	5.68	32.47	-0.144	细胞质Cytoplasm	无No
Dlo021675	DlASMT12	10	396	44.11	5.27	36.38	0.051	细胞质Cytoplasm	无No
Dlo021676	DlASMT13	10	163	18.04	4.80	35.40	-0.007	叶绿体Chloroplast	无No
Dlo027273	DlASMT14	13	502	55.16	6.22	35.67	-0.084	细胞质Cytoplasm	无No
Dlo027379	DlASMT15	13	361	40.58	5.66	22.65	-0.063	细胞质Cytoplasm	无No
Dlo029539	DlASMT16	14	359	39.66	5.64	41.86	-0.133	细胞质Cytoplasm	无No
Dlo032559	DlASMT17	15	370	41.19	5.79	32.94	-0.108	细胞质Cytoplasm	无No
Dlo032561	DlASMT18	15	374	41.28	5.32	32.59	-0.103	细胞质Cytoplasm	无No
Dlo010102	DlCOMT1	4	364	39.73	5.59	27.48	0.048	细胞质Cytoplasm	无No
Dlo010103	DlCOMT2	4	375	41.29	5.41	27.60	0.139	细胞质Cytoplasm	无No
Dlo021674	DlCOMT3	10	357	40.12	5.35	34.99	-0.173	细胞质Cytoplasm	无No
Dlo023091	DlCOMT4	11	357	39.29	5.93	43.69	0.018	细胞质Cytoplasm	无No
Dlo027274	DlCOMT5	13	351	38.88	5.43	48.29	0.027	细胞骨架Cytoskeleton	无No
Dlo027275	DlCOMT6	13	349	38.48	6.20	39.43	-0.080	细胞骨架Cytoskeleton	无No
Dlo027378	DlCOMT7	13	360	40.09	5.58	35.85	0.034	细胞质Cytoplasm	无No

基因ID Gene-ID	基因名称 Gene name	染色体定位 Chromosome location	氨基酸数 Amino acid number	分子量/kD Molecular weight	理论等电点 Theoretical pI	不稳定系数 The Instabi- lity index	亲水性 GRAVY	亚细胞定位 Subcellular location	信号肽 Signal peptide
Dlo018311	DlSNAT1	8	187	20.81	9.73	33.60	0.064	过氧化物酶体Peroxisome	无No
Dlo030488	DlSNAT2	14	225	24.68	4.89	44.33	-0.070	叶绿体Chloroplast	无No
Dlo004250	DlASMT1	2	347	38.76	5.22	39.04	0.077	细胞质Cytoplasm	无No
Dlo011828	DlASMT2	5	389	43.13	6.02	35.12	0.000	细胞质Cytoplasm	无No
Dlo011829	DlASMT3	5	442	49.48	5.77	41.16	-0.223	细胞质Cytoplasm	无No
基因ID Gene-ID	基因名称 Gene name	染色体定位 Chromosome location	氨基酸数 Amino acid number	分子量/kD Molecular weight	理论等电点 Theoretical pI	不稳定系数 The Instabi- Lity index	亲水性 GRAVY	亚细胞定位 Subcellular location	信号肽 Signal peptide
Dlo011833	DlASMT4	5	365	40.35	5.39	33.80	-0.099	细胞质Cytoplasm	无No
Dlo011834	DlASMT5	5	355	38.89	5.55	32.36	0.090	细胞质Cytoplasm	无No
Dlo011835	DlASMT6	5	354	39.07	5.55	35.08	0.007	细胞质Cytoplasm	无No
Dlo017622	DlASMT7	8	244	27.16	5.59	29.58	-0.029	细胞骨架Cytoskeleton	无No
Dlo017625	DlASMT8	8	367	41.17	5.20	36.62	-0.096	细胞骨架Cytoskeleton	无No
Dlo017687	DlASMT9	8	365	40.99	5.45	32.12	-0.074	细胞质Cytoplasm	无No
Dlo017690	DlASMT10	8	364	40.91	5.52	31.91	-0.074	叶绿体Chloroplast	无No
Dlo020696	DlASMT11	9	370	41.10	5.68	32.47	-0.144	细胞质Cytoplasm	无No
Dlo021675	DlASMT12	10	396	44.11	5.27	36.38	0.051	细胞质Cytoplasm	无No
Dlo021676	DlASMT13	10	163	18.04	4.80	35.40	-0.007	叶绿体Chloroplast	无No
Dlo027273	DlASMT14	13	502	55.16	6.22	35.67	-0.084	细胞质Cytoplasm	无No
Dlo027379	DlASMT15	13	361	40.58	5.66	22.65	-0.063	细胞质Cytoplasm	无No
Dlo029539	DlASMT16	14	359	39.66	5.64	41.86	-0.133	细胞质Cytoplasm	无No
Dlo032559	DlASMT17	15	370	41.19	5.79	32.94	-0.108	细胞质Cytoplasm	无No
Dlo032561	DlASMT18	15	374	41.28	5.32	32.59	-0.103	细胞质Cytoplasm	无No
Dlo010102	DlCOMT1	4	364	39.73	5.59	27.48	0.048	细胞质Cytoplasm	无No
Dlo010103	DlCOMT2	4	375	41.29	5.41	27.60	0.139	细胞质Cytoplasm	无No
Dlo021674	DlCOMT3	10	357	40.12	5.35	34.99	-0.173	细胞质Cytoplasm	无No
Dlo023091	DlCOMT4	11	357	39.29	5.93	43.69	0.018	细胞质Cytoplasm	无No
Dlo027274	DlCOMT5	13	351	38.88	5.43	48.29	0.027	细胞骨架Cytoskeleton	无No
Dlo027275	DlCOMT6	13	349	38.48	6.20	39.43	-0.080	细胞骨架Cytoskeleton	无No
Dlo027378	DlCOMT7	13	360	40.09	5.58	35.85	0.034	细胞质Cytoplasm	无No

基因Gene	P	Pearson相关系数 Pearson correlation
DlASMT4	0.029	0.999
DlASMT11	0.010	1.000
DlCOMT3	0.048	0.997

龙眼褪黑素合成途径SNAT、ASMT和COMT家族基因鉴定及表达分析

Whole-genome Identification and Expression Analysis of SNAT,ASMT and COMT Families of Melatonin Synthesis Pathway in Dimocarpus longan

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处理Treatment	P	Pearson相关系数 Pearson correlation
IAA	0.050	0.997
MeJA	0.045	0.997

处理Treatment	P	Pearson相关系数 Pearson correlation
IAA	0.050	0.997
MeJA	0.045	0.997

龙眼褪黑素合成途径SNAT、ASMT和COMT家族基因鉴定及表达分析

Whole-genome Identification and Expression Analysis of SNAT,ASMT and COMT Families of Melatonin Synthesis Pathway in Dimocarpus longan

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