草莓Metacaspase基因家族全基因组鉴定及表达分析

郑益平; 陈坚; 朱炳耀

doi:10.19303/j.issn.1008-0384.2020.11.003

草莓Metacaspase基因家族全基因组鉴定及表达分析

doi: 10.19303/j.issn.1008-0384.2020.11.003

福建省农业科学院生物技术研究所，福建　福州　350003

基金项目: 福建省科技计划公益类专项（2018R1019-10）

详细信息

作者简介:
郑益平（1988−），女，硕士，研究实习员，研究方向：花卉遗传育种（E-mail：360213443@qq.com）

通讯作者:
朱炳耀（1962−），男，副研究员，研究方向：植物资源收集与利用（E-mail：251112023@qq.com）

中图分类号: S 668.4
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出版历程
- 收稿日期: 2020-08-16
- 修回日期: 2020-10-08
- 网络出版日期: 2020-11-24
- 刊出日期: 2020-11-30

Genome-wide Identification and Expression Analysis of Metacaspase Gene Family in strawberry

Institute of Biotechnology, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350003, China

摘要

摘要: 目的精氨酸/赖氨酸特异性半胱氨酸酶（metacaspases，MCs）在植物生长发育的细胞程序性死亡（programmed cell death，PCD）中发挥重要的调控作用，为系统鉴定草莓FaMC基因家族，进行草莓FaMC基因家族全基因组鉴定及表达分析。方法通过草莓全基因组数据分析FaMC蛋白的理化性质、结构域、系统发育关系和保守基序，采用qRT-PCR技术对草莓6个组织器官和6个果实发育时期中的FaMC基因进行表达分析。结果蛋白序列分析表明，草莓基因组中21个FaMC成员分为3种类型：I型、I*型和II型。系统发育树和保守结构域分析表明：I型FaMC含N端前结构域且包含1个锌指结构；I*型FaMC含N端前结构域，但其中没有锌指结构；II型FaMC不含N端前结构域。基因表达分析发现，FaMC基因在不同组织中存在表达差异性，I型、I*型和II型FaMC基因分别在叶片、根和花中高表达。在果实发育过程中，多数I*型FaMC基因的表达水平呈现出高-低-高的动态变化趋势；部分I型、II型FaMC在果实发育过程中呈现相反的表达模式。结论 FaMC基因可能在草莓生长过程中发挥重要作用。
- 草莓 /
- metacaspase /
- 细胞程序性死亡（PCD） /
- 基因组 /
- 表达分析
Abstract: Objective To identify and study the genes in strawberry (Fragaria ananassa) which related to the synthesis of metacaspases (MCs), the enzymes that played vital roles in regulating the programmed cell death (PCD) in plants. Method The physiochemical properties, conserved domains, phylogenetic relationships and conserved motifs of FaMC proteins were analyzed based on strawberry whole genome data. The expressions of FaMC genes in 6 tissues and 6 fruit development stages of strawberry were carried out by qRT-PCR. Result The 21 FaMC proteins identified in this study could be divided into Type-I, Type-I*, and Type-II based on the differences on their sequences. The phylogenetic trees and conserved domains of the 3 different types showed the differentiations between them. Type-I FaMC protein consisted of N-terminal domain (NTD) with one zinc finger motif, Type-I* consisted of NTD but no zinc finger, and Type-II had not NTD. The FaMC genes were distinctively differently expressed in different tissues and developmental stages. Type-I expressed in the leaves, Type-I* in the roots, and Type-II in the flowers. And, during the berry development stage, most Type-I* exhibited a high-low-high U-pattern of changes, whereas, part of Type-I and Type-II had a reversed U-pattern. Conclusion FaMC genes were identified to be closely related to the synthesis of metacaspases that played important roles in the development and growth of strawberry plants.
- Strawberry /
- metacaspase /
- programmed cell death /
- genome /
- expression analysis

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图 1 草莓FaMC家族氨基酸序列比对分析

注：矩形：LSD1型锌指结构域；大粗实线：脯氨酸和谷氨酰胺的N端前结构域；中粗实线：p10亚基；细实线：p20亚基；虚线：连接区

Figure 1. Multiple sequence alignments of FaMC proteins in strawberry

Note: Rectangle: LSD1-type zinc finger motif; thickest solid line: Pro/Gln-rich NTD; thicker solid line: p10 subunit; solid line: p20 subunit; dotted line: linker.

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图 2 草莓与拟南芥MC家族蛋白质系统进化分析

注：拟南芥MC蛋白登录号：AtMC1（NP_171719.2）；AtMC2（NP_001031711.1）；AtMC3（NP_201229.1）；AtMC4（NP_178052.1）；AtMC5（NP_178051.1）；AtMC6（NP_178050.1）；AtMC7（NP_178049.2）；AtMC8（NP_173092.1）；AtMC9（NP_196040.1）

Figure 2. Phylogenetic tree of MC gene family in strawberry and Arabidopsis

Note: Accessions of AtMC proteins: AtMC1 (NP_171719.2); AtMC2 (NP_001031711.1); AtMC3 (NP_201229.1); AtMC4 (NP_178052.1); AtMC5 (NP_178051.1); AtMC6 (NP_178050.1); AtMC7 (NP_178049.2); AtMC8 (NP_173092.1); AtMC9 (NP_196040.1).

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图 3 草莓FaMC基因进化树、蛋白结构域及保守基序分布

注：A：基因进化树；B：蛋白结构域；C：保守基序

Figure 3. Distribution of phylogenetic trees, protein domains, and conserved motifs of FaMC genes in strawberry

Note: a: Phylogenetic tree; b: conserved domain; c: conserved motif.

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图 4 草莓FaMC基因的表达谱

注：A：FaMC基因在不同组织部位的表达谱；P1：叶片；P2：花；P3：根；P4：茎；P5：匍匐茎；P6：幼果。B：FaMC基因在果实不同发育时期的表达谱；S1：小绿期；S2：大绿期；S3：白果期；S4：始红期；S5：片红期；S6：全红期。红色代表高表达，绿色代表低表达。

Figure 4. Expression patterns of FaMC genes in strawberry

Note: a: Expression patterns of FaMC in different tissues; P1: leaf; P2: flower; P3: root; P4: stem; P5: stolon; P6: young fruit. b: expression patterns of FaMC in different fruit developmental stages; S1: small and green; S2: large and green; S3: white; S4: turning red; S5: partial red; S6: fully red. Red color represents high expression, green low expression.

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表 1 引物序列

Table 1. Primer sequences

基因 Gene	上游引物（5′→3′） Forward primer	下游引物（5′→3′） Reverse primer
FaActin	TGGGTTTGCTGGAGATGAT	CAGTTAGGAGAACTGGGTGC
FaMC1	ATGTTGGAGCACCTCACCTC	GCTTGACACCCACTCAGAAG
FaMC2	CAAAAACATCCGAAGGGCTA	CGGCAACCTTTCTTTAGCTG
FaMC3	CTCTCATCTTCCTCCTACAACG	GCATCGAGCTCCATATTCTTCC
FaMC4	TCTGGCAGTGCTGTTACGTC	CGCAGGCAGTTAATTGAGGC
FaMC5	CTTCCGTTCTCATGCTCACA	CAAAATCCGAGGGACACAGT
FaMC6	CGGTGTCAAGCTGCATTCTA	CCACCTTTTGTTCCTTTCCA
FaMC7	TCCCACTGACATGAACCTCA	GCTAGACTCACGCTGGTTCC
FaMC8	CGAGAAAGAGCAGATTGGACC	GCAAGTTGAGATCGAGTAGAGG
FaMC9	TGACCAGTATGTAAAGCCCG	ATCTCCTGAAGGTGTGGCAT
FaMC10	AATACGGAGCTCGATGGCTG	CGTTATGTTTGTCTTGGTAGGG
FaMC11	GAAATCCCTGAAATGGCTTG	GTGGGTGAACGATGGTTGA
FaMC12	ATTATCGTGGGGATGAAGTCG	CCATACATATTTTCCACCCCTG
FaMC13	GCCGCAATTGTTATGCCCAT	CACCCCTGTCCATTCTGCAA
FaMC14	CTTCACGATCTGCAACTCCA	GGCAGTTGGAGCAGTCTACC
FaMC15	CGAGTGGTCAGACAGCATCA	ACCAGCCGGAACTTTCTCGT
FaMC16	GCCCTTTCAATGCAGACCT	ACACCAACGAATCTCCAGCTT
FaMC17	ACAAACTCCAGAGCCACCAG	TGACAGCCACTGATGAGAATG
FaMC18	CGAGTTGTTGAAGCCGGA	TCTATTGCTTAGCGGAGCC
FaMC19	TACGGGAACATGATGACTGC	AGCTTTCCAAGACTCCCTCC
FaMC20	GCAGCAATTGTTAGTCCCAT	CACTTGTTCCTTTCCATACGC
FaMC21	GAAATACGGGTTCTGGTGGA	CTCGCAGGCAGTTAATTGGG

下载: 导出CSV

表 2 草莓FaMC蛋白理化性质

Table 2. Physiochemical properties and subcellular localization of FaMC proteins in strawberry

基因 Gene	基因组编号 Genome ID	氨基酸数 Amino acid	分子量 Molecular weight	等电点 pI	不稳定指数 Instability index	亲水性 Hydropathicity	亚细胞定位 Subcellular localization
FaMC1	augustus_masked-Fvb3-1-processed-gene-103.9	324	35555.30	5.43	37.04	−0.219	叶绿体 Chloroplast
FaMC2	maker-Fvb3-1-snap-gene-242.61	402	44269.43	5.06	42.48	−0.582	细胞质 Cytoplasmic
FaMC3	augustus_masked-Fvb3-1-processed-gene-281.5	326	36519.19	7.58	45.73	−0.110	叶绿体 Chloroplast
FaMC4	maker-Fvb7-2-augustus-gene-79.38	381	40904.39	6.42	41.60	−0.302	叶绿体 Chloroplast
FaMC5	maker-Fvb7-2-augustus-gene-253.66	358	38720.78	6.85	50.79	−0.327	细胞核 Nuclear
FaMC6	snap_masked-Fvb3-3-processed-gene-22.24	347	38805.38	8.14	47.98	−0.354	叶绿体 Chloroplast
FaMC7	maker-Fvb3-3-augustus-gene-63.33	413	45221.46	5.12	40.94	−0.616	细胞质 Cytoplasmic
FaMC8	maker-Fvb3-4-augustus-gene-109.35	323	35386.06	5.19	37.43	−0.207	叶绿体 Chloroplast
FaMC9	maker-Fvb3-4-augustus-gene-226.44	413	45224.44	5.06	40.97	−0.579	细胞质 Cytoplasmic
FaMC10	augustus_masked-Fvb3-4-processed-gene-255.9	290	31963.38	6.09	39.67	−0.362	叶绿体 Chloroplast
FaMC11	maker-Fvb5-1-augustus-gene-207.38	406	45451.82	8.95	52.83	−0.491	线粒体 Mitochondrial
FaMC12	maker-Fvb7-1-augustus-gene-86.37	381	40803.28	6.37	44.55	−0.282	叶绿体 Chloroplast
FaMC13	maker-Fvb7-1-augustus-gene-129.25	381	40807.37	6.48	45.56	−0.272	叶绿体 Chloroplast
FaMC14	maker-Fvb7-1-snap-gene-270.81	338	37729.50	9.10	62.14	−0.194	叶绿体 Chloroplast
FaMC15	maker-Fvb5-4-snap-gene-163.56	413	46277.63	8.88	54.06	−0.532	线粒体 Mitochondrial
FaMC16	snap_masked-Fvb3-2-processed-gene-39.23	350	39121.78	7.47	49.27	−0.334	叶绿体 Chloroplast
FaMC17	maker-Fvb3-2-augustus-gene-75.24	413	45214.42	5.06	41.30	−0.596	细胞质 Cytoplasmic
FaMC18	augustus_masked-Fvb3-2-processed-gene-201.8	323	35426.17	5.27	36.96	−0.203	线粒体 Mitochondrial
FaMC19	maker-Fvb7-3-augustus-gene-48.64	350	38180.67	7.59	39.34	−0.154	叶绿体 Chloroplast
FaMC20	maker-Fvb7-3-augustus-gene-157.36	371	39964.41	6.93	39.93	−0.267	线粒体 Mitochondrial
FaMC21	maker-Fvb7-4-augustus-gene-151.37	371	39772.15	6.34	40.74	−0.256	叶绿体 Chloroplast

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表 3 草莓FaMC蛋白氨基酸保守序列

Table 3. Conserved motifs of FaMC proteins in strawberry

Motif	长度/aa Length	氨基酸保守序列 Amino acid conserved sequence
motif1	50	PTKKNIRMALHWLVQGCQAGDSLVFHYSGHGTRQPNYTGDEVDGYDETLC
motif2	34	ELKGCINDAKCMKYLlinkerFKFPESSIRMLTEEE
motif3	50	PLDFETQGMIVDDEINAAIVRPJPAGVKLHAIVDACHSGTVLDLPFLCRM
motif4	29	PRSGVWKGTSGGEVISISGCDDBQTSADT
motif5	41	ITSTGAMTFAFIQAIERGHAATYGNJLNAMRSTIRNTGSGA
motif6	29	NHAPPPPPPNVHGRKRAVICGISYKYSRH
motif7	21	GLRQEPQLTASEEFDVYTKPF
motif8	50	AIQTILAETDGEITNQELVLRARKILKEQGYTQRPGLYCSDHHTDAPFVC
motif9	11	MLVGCSNCRTP
motif10	50	PTDMNLITDDDFRZFVDQLKKGCRLTIVSDSCHSGGLIDESVEQIGESHK
motif11	50	KTDIDVGKLRPTLFDVFGDDASPKVKKFMKVILNKLQSHEGEGSGGLMGK
motif12	50	IKNFLKQSAGDALKSRGIHVPSAFRRHGGDEEESEDREIDMGDGERGYMK
motif13	20	QLPPGAZSIRCALCQAVTLI
motif14	29	SAVASLLGGSSGAVTSLVGMLLTGGSVGG
motif15	11	RGGRYVWEDHR
motif16	50	DTLSGSCNKTKAIPFESILEHLTSLTGISTSDIATHFLELFAADASLKFR
motif17	21	GDPRALHSAPSSHEPPPPAPY
motif18	15	TPPGPPSYAYGALSY
motif19	11	GHPPPPPPRPY
motif20	15	NRELPLLALIFLLEQ

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