多毛番茄DCL、AGO和RDR基因家族全基因组鉴定及其对番茄褪绿病毒感染和非生物胁迫响应的分析

doi:10.7668/hbnxb.20195145

摘要/Abstract

摘要：

对多毛番茄全基因组的Dicer-like(DCL)、Argonaute(AGO)和RNA依赖性RNA聚合酶(RDR)基因家族进行生物信息学和表达模式分析,为深入研究DCL、AGO和RDR基因家族在多毛番茄响应非生物胁迫和病毒感染过程中的功能提供参考。以拟南芥DCL、AGO和RDR基因为参考序列,利用本地Perl语言和Pfam、SMART等软件检索多毛番茄LA1777基因组并确定多毛番茄DCL、AGO和RDR基因家族成员。通过ExPASy、GSDS 2.0、MEGA、Tbtools、SWISS-MODEL等工具对多毛番茄DCL、AGO和RDR家族基因进行生物信息学分析。根据非生物胁迫、番茄褪绿病毒(ToCV)处理和实时荧光定量PCR技术分析该类基因的表达模式。从多毛番茄中鉴定得到7个ShDCL、15个ShAGO和6个ShRDR基因,分别分布在第5,7,6条染色体上,其编码的蛋白与其他植物DCL、AGO和RDR结构相似,均含有该家族特有的保守结构域。系统发育分析表明,这些基因被分为4个亚组,与普通番茄之间具有较高的结构和功能相似性。ShDCL2a、ShDCL2c、ShDCL3、ShDCL4、ShAGO1b、ShAGO3、ShAGO4b、ShAGO5、ShAGO7、ShAGO10a、ShAGO10b、ShRDR1、ShRDR2、ShRDR3a、ShRDR6a和ShRDR6b在多种非生物胁迫和ToCV感染后显著上调表达,推测这些基因在非生物胁迫和病毒侵染过程中发挥着重要作用。

关键词: 多毛番茄, DCL, AGO, RDR, 非生物胁迫, 番茄褪绿病毒(ToCV)

Abstract:

Bioinformatics and expression patterns of Dicer-like(DCL),Argonaute(AGO)and RNA-dependent RNA polymerase(RDR)gene families in the whole genome of Solanum habrochaites were analyzed,so as to provide references for further study on the functions of DCL,AGO and RDR gene families in the response of S.habrochaites to abiotic and viral infection.Using Arabidopsis thaliana DCL,AGO and RDR genes as reference sequences,the genome of S.habrochaites LA1777 was searched by local perl language and software such as Pfam and SMART,and the members of ShDCL,ShAGO and ShRDR gene families were determined.Bioinformatics analysis of DCL,AGO and RDR family genes in S.habrochaites was carried out by means of ExPASy,GSDS 2.0,MEGA,Tbtools and SWISS-MODEL.According to abiotic stress treatment,Tomato chlorosis virus(ToCV)treatment and Real-time Fluorescence Quantitative PCR technology,the expression patterns of these genes were analyzed.Seven ShDCL,15 ShAGO and 6 ShRDR genes were identified from S.habrochaites,which were distributed on chromosome 5,7 and 6 respectively.The encoded proteins were similar in structure to DCL,AGO and RDR in other plants,and all of them contained conserved domains unique to this family.Phylogenetic analysis showed that these genes were divided into 4 subgroups,and there were high structural and functional similarities between S.habrochaites and S.lycopersicum.ShDCL2a,ShDCL2c,ShDCL3,ShDCL4,ShAGO1b,ShAGO3,ShAGO4b,ShAGO5,ShAGO7,ShAGO10a,ShAGO10b,ShRDR1,ShRDR2,ShRDR3a,ShRDR6a and ShRDR6b were significantly up-regulated after various abiotic stresses and ToCV infection.It is speculated that these genes play important roles in abiotic stress and virus infection.

Key words: Solanum habrochaites, DCL, AGO, RDR, Abiotic stress, Tomato chlorosis virus(ToCV)

康忱, 田哲娟, 高亢, 郝玲玉, 刘伟, 李亚栋, 吴志明. 多毛番茄DCL、AGO和RDR基因家族全基因组鉴定及其对番茄褪绿病毒感染和非生物胁迫响应的分析[J]. 华北农学报, 2024, 39(6): 1-17. doi: 10.7668/hbnxb.20195145.

KANG Chen, TIAN Zhejuan, GAO Kang, HAO Lingyu, LIU Wei, LI Yadong, WU Zhiming. Genome-wide Identification of Dicer-like,Argonaute and RNA-dependent RNA Polymerase Gene Families and Their Expression Analyses in Response to ToCV Infection and Abiotic Stresses in Solanum habrochaites[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 1-17. doi: 10.7668/hbnxb.20195145.

http://www.hbnxb.net/CN/Y2024/V39/I6/1

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引物名称 Gene name	正向引物(5'—3') Forward sequence (5'—3')
Actin	CAGCACCTTCCAGCAGATGT	AGAAGTCTTTTCCTTTCACCAAC
ShDCL1	GTTTTCCTCCCTCCTGAGCT	GCGGTCAATGCTTCCAAGAT
ShDCL2a	TGCTTCCGGAACTATGTGGT	AGCTGAGGGATCAAACCGAA
ShDCL2b	GCTCGCAAAATACCGGGATT	TGCCTCAACCACATCAGCTA
ShDCL2c	CTTCAACACTCTTGCCTGCA	CCGGTTAAAGTAGCAGTGCC
ShDCL2d	CGAAGAGCTCCTAACGTCCT	TGATGTCCACTCCAAGCCAT
ShDCL3	TGTGTGGCTAAGCAAGGAGA	GCTCTCTGCGGAAAGAATGG
ShDCL4	TTGGAAGTGCGGGTTTCTTG	GCACGCCCTCTAGACTGTAT
ShAGO1a	CGGCCTGGAAAGGGTAGTAA	TTACCAGCTGCTCCATGACA
ShAGO1b	GAAAGTACCGCATCGCCAAT	ACCCTCGACAATCTTGCAGA
ShAGO2a	TCGTCGTACAAGCCAGAAGT	TTGAACTGAATCTCGCGCTG
ShAGO2b	TCCCGGGATGCTGACAAATA	GACTCCCACTCACACCATCA
ShAGO3	ACAACCTCCCTTGAACCCAA	GCCTTCCACCACAGATTTCC
ShAGO4a	AAATGCTCTGCGTGGTCAAG	CCAGCACCGACATCAACAAA
ShAGO4b	ACAACGGTACCAATGGCAAC	CTCAGACTCTTGACCTCGCA
ShAGO4c	TTGAGTTGGAGGTGGGTGTT	TTTTGCCTTCGCCCAATCAA
ShAGO4d	TCGCCTACTCACCAACCATT	TCTTTGCCAGCTAGCTCCAT
ShAGO5	CTAAATGGCAGGCAGGTCAC	GGTGGGATCTGTTGGGAGAA
ShAGO6	GCAGAGCCAGAGAGAGTTGA	ACGGCCCTCTTTTCATCAGA
ShAGO7	TAGATGTTGTGTTGCGCGAG	TCTCGTGGAAAGCAGTCACT
ShAGO10a	AAGGCCTTGTACAGCAGCTA	TCCCAAGTTGCCCATAACCT
ShAGO10b	TTGCAGACCATTCGCCAAAA	AAGTTGATGCAAGCCCAGTG
ShAGO15	GCCAATCGTACTTCCCCAAC	TTTTCGCCTTTGTCCAGTCG
ShRDR1	TTCGGCTTGGTACCACGTTA	TGAGAACTGGCCTGTTACGT
ShRDR2	CGAGAGGCCATGGTAGAGTT	CCCAAGTCTCCAAAACCTGC
ShRDR3a	ACTTGCTGTCCCTGTGAAGA	GATTCGGCTCCACGGTTTAC
ShRDR3b	ACACGGGCCATGAACTTAGA	CGCAGAACCAGCTACTTTCC
ShRDR6a	TGCTAGGCGTGATGAACTCT	CCCTCCAACCAACCACAAAG
ShRDR6b	TCCCCAGAAAACTGCCATCT	CTGAACCTCCCCATCCAGTT

引物名称 Gene name	正向引物(5'—3') Forward sequence (5'—3')
Actin	CAGCACCTTCCAGCAGATGT	AGAAGTCTTTTCCTTTCACCAAC
ShDCL1	GTTTTCCTCCCTCCTGAGCT	GCGGTCAATGCTTCCAAGAT
ShDCL2a	TGCTTCCGGAACTATGTGGT	AGCTGAGGGATCAAACCGAA
ShDCL2b	GCTCGCAAAATACCGGGATT	TGCCTCAACCACATCAGCTA
ShDCL2c	CTTCAACACTCTTGCCTGCA	CCGGTTAAAGTAGCAGTGCC
ShDCL2d	CGAAGAGCTCCTAACGTCCT	TGATGTCCACTCCAAGCCAT
ShDCL3	TGTGTGGCTAAGCAAGGAGA	GCTCTCTGCGGAAAGAATGG
ShDCL4	TTGGAAGTGCGGGTTTCTTG	GCACGCCCTCTAGACTGTAT
ShAGO1a	CGGCCTGGAAAGGGTAGTAA	TTACCAGCTGCTCCATGACA
ShAGO1b	GAAAGTACCGCATCGCCAAT	ACCCTCGACAATCTTGCAGA
ShAGO2a	TCGTCGTACAAGCCAGAAGT	TTGAACTGAATCTCGCGCTG
ShAGO2b	TCCCGGGATGCTGACAAATA	GACTCCCACTCACACCATCA
ShAGO3	ACAACCTCCCTTGAACCCAA	GCCTTCCACCACAGATTTCC
ShAGO4a	AAATGCTCTGCGTGGTCAAG	CCAGCACCGACATCAACAAA
ShAGO4b	ACAACGGTACCAATGGCAAC	CTCAGACTCTTGACCTCGCA
ShAGO4c	TTGAGTTGGAGGTGGGTGTT	TTTTGCCTTCGCCCAATCAA
ShAGO4d	TCGCCTACTCACCAACCATT	TCTTTGCCAGCTAGCTCCAT
ShAGO5	CTAAATGGCAGGCAGGTCAC	GGTGGGATCTGTTGGGAGAA
ShAGO6	GCAGAGCCAGAGAGAGTTGA	ACGGCCCTCTTTTCATCAGA
ShAGO7	TAGATGTTGTGTTGCGCGAG	TCTCGTGGAAAGCAGTCACT
ShAGO10a	AAGGCCTTGTACAGCAGCTA	TCCCAAGTTGCCCATAACCT
ShAGO10b	TTGCAGACCATTCGCCAAAA	AAGTTGATGCAAGCCCAGTG
ShAGO15	GCCAATCGTACTTCCCCAAC	TTTTCGCCTTTGTCCAGTCG
ShRDR1	TTCGGCTTGGTACCACGTTA	TGAGAACTGGCCTGTTACGT
ShRDR2	CGAGAGGCCATGGTAGAGTT	CCCAAGTCTCCAAAACCTGC
ShRDR3a	ACTTGCTGTCCCTGTGAAGA	GATTCGGCTCCACGGTTTAC
ShRDR3b	ACACGGGCCATGAACTTAGA	CGCAGAACCAGCTACTTTCC
ShRDR6a	TGCTAGGCGTGATGAACTCT	CCCTCCAACCAACCACAAAG
ShRDR6b	TCCCCAGAAAACTGCCATCT	CTGAACCTCCCCATCCAGTT

序号 Number	基因名称 Gene name	序列登录号 Gene ID	位置/bp Genomic location	CDS长度/ bp CDS length	氨基酸长度/个 Amino acid length	等电点 PI	分子质量/ku Molecular weight	亚细胞定位 Subcellular localization	同源性最高的番茄基因编号 Tomato gene number with the highest homology
Dicer-like(DCL)
1	ShDCL1	Shab10g000160.1	chr10:117674—131266	5 043	1 680	5.69	188.69	1	Solyc10g005130
2	ShDCL2a	Shab06g007210.1	chr6:31381067—31393838	4 209	1 402	6.53	158.02	1	Solyc06g048960
3	ShDCL2b	Shab11g003360.1	chr11:2797006—2801926	3 531	1 176	7.45	132.99	1	Solyc11g008540
4	ShDCL2c	Shab11g003340.1	chr11:2779197—2786011	1 083	360	4.81	39.51	1	Solyc11g008520
5	ShDCL2d	Shab11g003370.1	chr11:2805943—2811457	3 426	1 141	7.75	129.65	1	Solyc11g008530
6	ShDCL3	Shab08g011760.1	chr8:52079657—52106758	3 843	1 280	7.66	143.50	1	Solyc08g067210
7	ShDCL4	Shab07g000040.1	chr7:61116—94001	4 862	1 620	6.47	182.25	1	Solyc07g005030
Argonaute(AGO)
1	ShAGO1a	Shab06g018510.1	chr6:46211483—46220383	3 165	1 054	9.48	116.70	1	Solyc06g072300
2	ShAGO1b	Shab03g017920.1	chr3:56608587—56615954	3 459	1 152	9.46	126.91	1	Solyc03g098280
3	ShAGO2a	Shab02g010500.1	chr2:41010367—41017103	3 120	1 039	9.39	115.62	1	Solyc02g069260
4	ShAGO2b	Shab02g010510.1	chr2:41030311—41034222	2 946	981	9.02	110.74	1	Solyc02g069270
5	ShAGO3	Shab02g010520.1	chr2:41036447—41040263	3 000	999	8.90	112.47	1	Solyc02g069280
6	ShAGO4a	Shab01g003450.1	chr1:3233010—3238637	2 604	867	9.03	97.20	1,2	Solyc01g008960
7	ShAGO4b	Shab06g019700.1	chr6:47008379—47013982	2 769	922	9.33	103.56	1	Solyc06g073540
8	ShAGO4c	Shab06g019690.1	chr6:46997111—47003903	2 748	915	9.31	102.46	3	Solyc06g073530
9	ShAGO4d	Shab01g021530.1	chr1:86969262—86973908	2 646	881	9.23	98.86	1	Solyc01g096750
10	ShAGO5	Shab06g020800.1	chr6:47999599—48006297	2 775	924	9.61	102.99	1	Solyc06g074730
11	ShAGO6	Shab07g013570.1	chr7:62215729—62224152	2 574	857	8.75	96.62	1,2,3	Solyc07g049500
12	ShAGO7	Shab01g005290.1	chr1:7642775—7651741	2 934	977	9.31	111.06	1,2	Solyc01g010970
13	ShAGO10a	Shab09g017290.2	chr73523970—73531554	3 045	1 014	9.23	114.50	1	Solyc09g082830
14	ShAGO10b	Shab12g001730.1	chr12:1289252—1303393	2 817	938	9.31	106.26	1,2	Solyc12g006790
15	ShAGO15	Shab03g020040.1	chr3:58631540—58637276	2 808	935	9.55	106.33	2,3	Solyc03g111760
RNA-dependent RNA polymerase(RDR)
1	ShRDR1	Shab05g002220.1	chr5:2080655—2087989	3 204	1 067	8.56	121.10	3	Solyc05g007510
2	ShRDR2	Shab03g022350.1	chr3:60533575—60539371	3 360	1 119	6.64	127.44	1	Solyc03g114140
3	ShRDR3a	Shab12g002370.1	chr12:1956348—1968345	3 156	1 051	8.06	121.03	3,4	Solyc12g008410
4	ShRDR3b	Shab06g008130.1	chr6:34277469—34305043	3 042	1 013	7.80	115.50	3	Solyc06g051170
5	ShRDR6a	Shab04g006050	chr4:5359143—5366807	3 594	1 197	6.47	136.17	3	Solyc04g014870
6	ShRDR6b	Shab08g014840.1	chr8:56984431—56986296	1 866	621	8.76	70.50	3	Solyc08g075820

序号 Number	基因名称 Gene name	序列登录号 Gene ID	位置/bp Genomic location	CDS长度/ bp CDS length	氨基酸长度/个 Amino acid length	等电点 PI	分子质量/ku Molecular weight	亚细胞定位 Subcellular localization	同源性最高的番茄基因编号 Tomato gene number with the highest homology
Dicer-like(DCL)
1	ShDCL1	Shab10g000160.1	chr10:117674—131266	5 043	1 680	5.69	188.69	1	Solyc10g005130
2	ShDCL2a	Shab06g007210.1	chr6:31381067—31393838	4 209	1 402	6.53	158.02	1	Solyc06g048960
3	ShDCL2b	Shab11g003360.1	chr11:2797006—2801926	3 531	1 176	7.45	132.99	1	Solyc11g008540
4	ShDCL2c	Shab11g003340.1	chr11:2779197—2786011	1 083	360	4.81	39.51	1	Solyc11g008520
5	ShDCL2d	Shab11g003370.1	chr11:2805943—2811457	3 426	1 141	7.75	129.65	1	Solyc11g008530
6	ShDCL3	Shab08g011760.1	chr8:52079657—52106758	3 843	1 280	7.66	143.50	1	Solyc08g067210
7	ShDCL4	Shab07g000040.1	chr7:61116—94001	4 862	1 620	6.47	182.25	1	Solyc07g005030
Argonaute(AGO)
1	ShAGO1a	Shab06g018510.1	chr6:46211483—46220383	3 165	1 054	9.48	116.70	1	Solyc06g072300
2	ShAGO1b	Shab03g017920.1	chr3:56608587—56615954	3 459	1 152	9.46	126.91	1	Solyc03g098280
3	ShAGO2a	Shab02g010500.1	chr2:41010367—41017103	3 120	1 039	9.39	115.62	1	Solyc02g069260
4	ShAGO2b	Shab02g010510.1	chr2:41030311—41034222	2 946	981	9.02	110.74	1	Solyc02g069270
5	ShAGO3	Shab02g010520.1	chr2:41036447—41040263	3 000	999	8.90	112.47	1	Solyc02g069280
6	ShAGO4a	Shab01g003450.1	chr1:3233010—3238637	2 604	867	9.03	97.20	1,2	Solyc01g008960
7	ShAGO4b	Shab06g019700.1	chr6:47008379—47013982	2 769	922	9.33	103.56	1	Solyc06g073540
8	ShAGO4c	Shab06g019690.1	chr6:46997111—47003903	2 748	915	9.31	102.46	3	Solyc06g073530
9	ShAGO4d	Shab01g021530.1	chr1:86969262—86973908	2 646	881	9.23	98.86	1	Solyc01g096750
10	ShAGO5	Shab06g020800.1	chr6:47999599—48006297	2 775	924	9.61	102.99	1	Solyc06g074730
11	ShAGO6	Shab07g013570.1	chr7:62215729—62224152	2 574	857	8.75	96.62	1,2,3	Solyc07g049500
12	ShAGO7	Shab01g005290.1	chr1:7642775—7651741	2 934	977	9.31	111.06	1,2	Solyc01g010970
13	ShAGO10a	Shab09g017290.2	chr73523970—73531554	3 045	1 014	9.23	114.50	1	Solyc09g082830
14	ShAGO10b	Shab12g001730.1	chr12:1289252—1303393	2 817	938	9.31	106.26	1,2	Solyc12g006790
15	ShAGO15	Shab03g020040.1	chr3:58631540—58637276	2 808	935	9.55	106.33	2,3	Solyc03g111760
RNA-dependent RNA polymerase(RDR)
1	ShRDR1	Shab05g002220.1	chr5:2080655—2087989	3 204	1 067	8.56	121.10	3	Solyc05g007510
2	ShRDR2	Shab03g022350.1	chr3:60533575—60539371	3 360	1 119	6.64	127.44	1	Solyc03g114140
3	ShRDR3a	Shab12g002370.1	chr12:1956348—1968345	3 156	1 051	8.06	121.03	3,4	Solyc12g008410
4	ShRDR3b	Shab06g008130.1	chr6:34277469—34305043	3 042	1 013	7.80	115.50	3	Solyc06g051170
5	ShRDR6a	Shab04g006050	chr4:5359143—5366807	3 594	1 197	6.47	136.17	3	Solyc04g014870
6	ShRDR6b	Shab08g014840.1	chr8:56984431—56986296	1 866	621	8.76	70.50	3	Solyc08g075820

多毛番茄 S.habrochaites		番茄 S.lycopersicum		拟南芥 Arabidopsis
AGO	Motifs	AGO	Motifs	AGO	Motifs
ShAGO2a	DDD/H	SlAGO2a	DDD/H	AtAGO2	DDD/H
ShAGO2b	DDD/H	ShAGO2b	DDD/H	AtAGO3	DDD/H
ShAGO3	DDD/S	ShAGO3	DDD/S	AtAGO4	DDH/S
ShAGO4a	DDH/P	ShAGO4a	DDH/P	AtAGO6	DDH/P
ShAGO4b	DDH/P	ShAGO4b	DDH/P	AtAGO8	DDH/P
ShAGO4c	DDH/P	ShAGO4c	DDH/P	AtAGO9	DDH/R
ShAGO4d	DDH/P	ShAGO4d	DDH/P
ShAGO5	DDH/P	ShAGO6	DDH/P
ShAGO6	DDH/P	ShAGO15	GQR/P
ShAGO15	GQR/P

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