草莓LIM家族候选基因的鉴定及在非生物胁迫下的表达

doi:10.16420/j.issn.0513-353x.2020-0595

摘要/Abstract

摘要：

利用NCBI、GDR数据库对草莓LIM家族候选基因在草莓基因组水平进行生物信息学分析,鉴定获得4个亚族37个候选LIM基因,其中9个来自森林草莓（Fragaria vesca L.）,28个来自凤梨草莓（F. ananassa Duch.）。草莓LIM家族候选基因主要分布在细胞核;其密码子偏好性较弱,偏向使用以A/T结尾的同义密码子。FvLIM和FaLIM启动子序列上游2 kb区域包含大量激素及非生物胁迫响应元件。10% PEG、200 mmol · L^-1 NaCl、4 ℃低温胁迫下,随着处理时间延长LIM家族候选基因在凤梨草莓‘红颜’中比森林草莓中上调趋势更明显。4 ℃低温处理下,森林草莓中以FvLIM5（12 h）和FvLIM7（24 h）上调最为明显,而凤梨草莓‘红颜’中以FaLIM25（12 h）和FaLIM18（24 h）上调最为显著,总体对低温响应较明显。实时荧光定量PCR分析结果表明,草莓LIM家族候选基因在不同组织中的表达量存在一定差异,在根和花中的表达量偏高。综合以上分析结果,预测草莓LIM家族候选基因可能在响应非生物胁迫以及根和花的发育中发挥重要作用。

关键词: 草莓, LIM家族, 生物信息学, qRT-PCR

Abstract:

In this paper,NCBI and GDR database were used to analyze the candidate genes of LIM family in strawberry genome,and 37 LIM genes could be divided into four subgroups,nine LIM genes were identified from Fragaria vescaL. and 28 LIM genes were identified from cultivated strawberry（F. ananassaDuch.）. The LIM family candidate genes were mainly expressed in the nucleus. Their codon bias were less preferred and tended to use the synonymous codon ending in A/T. The upstream 2 kb region of promoter sequences of FvLIM and FaLIM contained a large number of hormone response elements and abiotic stress elements. The expression levels of LIM family candidate genes under 200 mmol · L^-1 NaCl,10% PEG,and 4 ℃ low temperature,FaLIMwere more significantly up-regulated than FvLIM with the treatment time. Under 4 ℃ low temperature treatment,FvLIM5（12 h）and FvLIM7（24 h）were significantly up-regulated in F. vesca,FaLIM25（12 h）and FaLIM18（24 h）significantly up-regulated in F. ananassa‘Benihoppe’,the overall response to low temperature is obvious. The results of qRT-PCR showed that most of LIM family candidate genes were highly expressed in roots and flowers,their diverse and specific expression could be detected in five tissues. Based on the above analysis results,it was speculated that the LIM family candidate genes of strawberry might play an important role in abiotic stress and in roots and flowers development.

Key words: strawberry, LIM gene family, bioinformatics, qRT-PCR

中图分类号:

S668.4

张志强, 卢世雄, 马宗桓, 李彦彪, 高彩霞, 陈佰鸿, 毛娟. 草莓LIM家族候选基因的鉴定及在非生物胁迫下的表达[J]. 园艺学报, 2021, 48(8): 1485-1503.

ZHANG Zhiqiang, LU Shixiong, MA Zonghuan, LI Yanbiao, GAO Caixia, CHEN Baihong, MAO Juan. Bioinformatic Identification and Expression Analysis of Candidate Genes of LIM Protein Family in Strawberry Under Abiotic Stresses[J]. Acta Horticulturae Sinica, 2021, 48(8): 1485-1503.

图/表 14

参考文献 43

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基因 Gene	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer
FvLIM1	ATATGTGGCTTGACGCAGAGACG	GAGGACGCTGAGGAGGAAGAGG
FvLIM2	AAACACCACCACATCCAGCTCTTC	GGCTTGCTCGTTCGTTGATTGC
FvLIM3	ATATGTGGCTTGACGCAGAGACG	GAGGACGCTGAGGAGGAAGAGG
FvLIM4	GCACCACCACACCCAACTCATC	GCAACTGCTGTGGCTTTCTCATTG
FvLIM5	TTGCCCAGTTGTTCAAGGAGAAGG	TGCTGCGTCTGGCTTTGGTTC
FvLIM6	TGCAAGGCATGTGAGAAGACTGTG	AGTGGTGGCATCTGAAGCAAGC
FvLIM7	GAATAGGGCACCCAGCAAACTCTC	TGAGCACACCTGAAGCATGACTTG
FvLIM8	TTAGACCATGCAATCGCGCTCTC	GCTCGTTATCCTTCTCCTGCCATC
FvLIM9	CTGGTATGTTTTCTGGCACGCAAG	TGGCTCTCCACTGTCACCTTCTC
FaLIM1	ATATGTGGCTTGACGCAGAGACG	AAGAGGACGCTGAGGAGGAAGAC
FaLIM2	GCCGAAGATTGGAGCAGATTACCG	ACGAGAATTGCCGTCACTTCACAG
FaLIM3	ATATGTGGCTTGACGCAGAGACG	GAGGACGCTGAGGAGGAAGAGG
FaLIM4	ATATGTGGCTTGACGCAGAGACG	AGGACGCTGAGAAGGAAGAGGAAG
FaLIM5	GCACCACCACACCCAACTCATC	GCAACTGCTGTGGCTTTCTCATTG
FaLIM6	GATAGGACTCCACGGTGTTGTAGC	TTCAAGGTAAAGGGGCTGGCATTC
FaLIM7	GCTAGATGCCGAGCTTATGTCTGG	GCGTGCCCTTCTTTGATGAATTGG
FaLIM8	AAACACCACCACATCCAGCTCTTC	GGCTTGCTCGTTCGTTGATTGC
FaLIM9	TTGCCCAGTTGTTCAAGGAGAAGG	TGCTGCGTCTGGCTTTGGTTG
FaLIM10	TTGCCCAGTTGTTCAAGGAGAAGG	TGCTGCGTCTGGCTTTGGTTC
FaLIM11	CACCGCCTCTACTGTAAGCATCAC	TACTCCGCCACTGTGCTCTCAG
FaLIM12	TTGCCCAGTTGTTCAAGGAGAAGG	TGCTGCGTCTGGCTTTGGTTC
FaLIM13	TGCAAGGCATGTGAGAAGACTGTG	AGTGGTGGCATCTGAAGCAAGC
FaLIM14	GAATAGGGCACCCAGCAAACTCTC	TGAGCACACCTGAAGCATGACTTG
FaLIM15	AGTGCTTCTGTTGTCGCTCTTGTC	GGATGGGCCAGCTCCTTGAAAC
FaLIM16	AGGAGAAGGGGAGCTACAACCATC	TGGAATGCTGGCAACTGCTACTG
FaLIM17	GAATAGGGCACCCAGCAAACTCTC	TGAGCACACCTGAAGCATGACTTG
FaLIM18	GAATAGGGCACCCAGCAAACTCTC	TGAGCACACCTGAAGCATGACTTG
FaLIM19	TTAGACCATGCAATCGCGCTCTC	GCTCGTTATCCTTCTCCTGCCATC
FaLIM20	CGACTAGGTCACCCAGCAAAGC	CGACTTGTGATATGCCTGGCTCTC
FaLIM21	TTAGACCATGCAATCGCGCTCTC	GCTCGTTATCCTTCTCCTGCCATC
FaLIM22	TCAAGGAGAAGGGGAGCTACAACC	TTAGGCTTCTGGAATGCTGGCAAC
FaLIM23	TCAAGGAGAAGGGGAGCTACAACC	TTAGGCTTCTGGAATGCTGGCAAC
FaLIM24	AGTGCTTCTGTTGTCGCTCTTGTC	GGATGGGCCAGCTCCTTGAAAC
FaLIM25	AAGGTCCATTTCGCTCAGCTCTTC	CTTCTGGTTGTGGCTCTGCTTCTG
FaLIM26	CTGGTATGTTTTCTGGCACGCAAG	TGGCTCTCCACTGTCACCTTCTC
FaLIM27	AGTGCTTCTGTTGTCGCTCTTGTG	GGATGGGCCAGCTCCTTGAAAC
FaLIM28	GAATAGGGCACCCAGCAAACTCTC	TGAGCACACCTGAAGCATGACTTG
GAPDH	CATTCATCACCACCGACTACA	GAAGGGTCTTCTCATCCTTGAC

基因 Gene	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer
FvLIM1	ATATGTGGCTTGACGCAGAGACG	GAGGACGCTGAGGAGGAAGAGG
FvLIM2	AAACACCACCACATCCAGCTCTTC	GGCTTGCTCGTTCGTTGATTGC
FvLIM3	ATATGTGGCTTGACGCAGAGACG	GAGGACGCTGAGGAGGAAGAGG
FvLIM4	GCACCACCACACCCAACTCATC	GCAACTGCTGTGGCTTTCTCATTG
FvLIM5	TTGCCCAGTTGTTCAAGGAGAAGG	TGCTGCGTCTGGCTTTGGTTC
FvLIM6	TGCAAGGCATGTGAGAAGACTGTG	AGTGGTGGCATCTGAAGCAAGC
FvLIM7	GAATAGGGCACCCAGCAAACTCTC	TGAGCACACCTGAAGCATGACTTG
FvLIM8	TTAGACCATGCAATCGCGCTCTC	GCTCGTTATCCTTCTCCTGCCATC
FvLIM9	CTGGTATGTTTTCTGGCACGCAAG	TGGCTCTCCACTGTCACCTTCTC
FaLIM1	ATATGTGGCTTGACGCAGAGACG	AAGAGGACGCTGAGGAGGAAGAC
FaLIM2	GCCGAAGATTGGAGCAGATTACCG	ACGAGAATTGCCGTCACTTCACAG
FaLIM3	ATATGTGGCTTGACGCAGAGACG	GAGGACGCTGAGGAGGAAGAGG
FaLIM4	ATATGTGGCTTGACGCAGAGACG	AGGACGCTGAGAAGGAAGAGGAAG
FaLIM5	GCACCACCACACCCAACTCATC	GCAACTGCTGTGGCTTTCTCATTG
FaLIM6	GATAGGACTCCACGGTGTTGTAGC	TTCAAGGTAAAGGGGCTGGCATTC
FaLIM7	GCTAGATGCCGAGCTTATGTCTGG	GCGTGCCCTTCTTTGATGAATTGG
FaLIM8	AAACACCACCACATCCAGCTCTTC	GGCTTGCTCGTTCGTTGATTGC
FaLIM9	TTGCCCAGTTGTTCAAGGAGAAGG	TGCTGCGTCTGGCTTTGGTTG
FaLIM10	TTGCCCAGTTGTTCAAGGAGAAGG	TGCTGCGTCTGGCTTTGGTTC
FaLIM11	CACCGCCTCTACTGTAAGCATCAC	TACTCCGCCACTGTGCTCTCAG
FaLIM12	TTGCCCAGTTGTTCAAGGAGAAGG	TGCTGCGTCTGGCTTTGGTTC
FaLIM13	TGCAAGGCATGTGAGAAGACTGTG	AGTGGTGGCATCTGAAGCAAGC
FaLIM14	GAATAGGGCACCCAGCAAACTCTC	TGAGCACACCTGAAGCATGACTTG
FaLIM15	AGTGCTTCTGTTGTCGCTCTTGTC	GGATGGGCCAGCTCCTTGAAAC
FaLIM16	AGGAGAAGGGGAGCTACAACCATC	TGGAATGCTGGCAACTGCTACTG
FaLIM17	GAATAGGGCACCCAGCAAACTCTC	TGAGCACACCTGAAGCATGACTTG
FaLIM18	GAATAGGGCACCCAGCAAACTCTC	TGAGCACACCTGAAGCATGACTTG
FaLIM19	TTAGACCATGCAATCGCGCTCTC	GCTCGTTATCCTTCTCCTGCCATC
FaLIM20	CGACTAGGTCACCCAGCAAAGC	CGACTTGTGATATGCCTGGCTCTC
FaLIM21	TTAGACCATGCAATCGCGCTCTC	GCTCGTTATCCTTCTCCTGCCATC
FaLIM22	TCAAGGAGAAGGGGAGCTACAACC	TTAGGCTTCTGGAATGCTGGCAAC
FaLIM23	TCAAGGAGAAGGGGAGCTACAACC	TTAGGCTTCTGGAATGCTGGCAAC
FaLIM24	AGTGCTTCTGTTGTCGCTCTTGTC	GGATGGGCCAGCTCCTTGAAAC
FaLIM25	AAGGTCCATTTCGCTCAGCTCTTC	CTTCTGGTTGTGGCTCTGCTTCTG
FaLIM26	CTGGTATGTTTTCTGGCACGCAAG	TGGCTCTCCACTGTCACCTTCTC
FaLIM27	AGTGCTTCTGTTGTCGCTCTTGTG	GGATGGGCCAGCTCCTTGAAAC
FaLIM28	GAATAGGGCACCCAGCAAACTCTC	TGAGCACACCTGAAGCATGACTTG
GAPDH	CATTCATCACCACCGACTACA	GAAGGGTCTTCTCATCCTTGAC

名称 Name	染色体定位 Chromosome location	氨基酸数 Amino acids	分子量 Molecular weight	等电点 pI	不稳定指数 Instability index	亲水性 Hydrophilic	亚细胞定位 Subcellular localization
FvLIM1	Fvb1:7945940..7952121	538	60 510.36	5.20	53.78	-0.718	细胞核 Nucleus
FvLIM2	Fvb2:2982770..2998244	197	22 232.22	8.89	42.90	-0.703	细胞核 Nucleus
FvLIM3	Fvb2:5932861..5935114	527	59 323.18	5.33	52.19	-0.698	细胞核 Nucleus
FvLIM4	Fvb2:24843809..24848493	191	21 205.29	9.05	29.01	-0.539	细胞核 Nucleus
FvLIM5	Fvb5:1235177..1237221	212	23 391.44	7.52	45.56	-0.62	细胞核 Nucleus
FvLIM6	Fvb5:9381785..9385122	188	21 211.15	8.86	28.77	-0.586	细胞核 Nucleus
FvLIM7	Fvb7:12341249..12343536	217	23 962.13	6.50	45.65	-0.567	细胞核 Nucleus
FvLIM8	Fvb7:18342947..18346948	504	57 014.81	8.26	52.67	-0.541	细胞核Nucleus
FvLIM9	Fvb7:18517083..18519868	200	21 721.91	9.08	37.48	-0.362	细胞核 Nucleus
FaLIM1	Fvb1-1:20431547..20437334	534	59 915.92	5.22	52.12	-0.668	细胞核 Nucleus
FaLIM2	Fvb1-2:9164763..9171264	533	60 168.24	5.39	51.21	-0.737	细胞核 Nucleus
FaLIM3	Fvb1-3:8145568..8151824	539	60 522.40	5.20	54.39	-0.719	细胞核 Nucleus
FaLIM4	Fvb1-4:6945308..6951056	538	60 571.45	5.17	53.06	-0.712	细胞核 Nucleus
FaLIM5	Fvb2-2:728488..730564	191	21 205.29	9.05	29.01	-0.539	细胞核 Nucleus
FaLIM6	Fvb2-2:18290991..18293017	231	27 087.33	7.80	45.08	-0.512	细胞核 Nucleus
FaLIM7	Fvb2-3:2848239..2854332	491	55 136.60	5.75	44.43	-0.671	细胞核 Nucleus
FaLIM8	Fvb2-4:1747334..1750087	176	19 899.54	8.83	44.58	-0.618	细胞核 Nucleus
FaLIM9	Fvb5-1:1118664..1120389	213	23 551.67	7.92	45.67	-0.623	细胞核 Nucleus
FaLIM10	Fvb5-2:1037693..1039462	216	23 835.04	8.18	44.28	-0.569	细胞核 Nucleus
FaLIM11	Fvb5-3:18813760..18816438	188	21 172.07	8.86	31.15	-0.599	细胞核 Nucleus
FaLIM12	Fvb5-4:1027068..1028886	211	23 297.40	7.52	43.54	-0.607	细胞核 Nucleus
FaLIM13	Fvb5-4:7252377..7255038	188	21 199.10	8.86	29.68	-0.613	细胞核 Nucleus
FaLIM14	Fvb7-1:16915680..16917768	217	23 964.10	6.50	45.95	-0.579	细胞核 Nucleus
FaLIM15	Fvb7-1:25526059..25530050	506	57 362.23	8.54	52.15	-0.549	细胞核Nucleus
FaLIM16	Fvb7-1:25665993..25670535	200	21 818.02	9.08	37.58	-0.360	细胞核 Nucleus
FaLIM17	Fvb7-2:11429297..11431530	217	23 962.13	6.50	45.65	-0.567	细胞核 Nucleus
FaLIM18	Fvb7-2:17869656..17871925	217	23 949.13	6.50	45.65	-0.554	细胞核 Nucleus
FaLIM19	Fvb7-2:23292270..23296225	493	55 877.59	8.37	53.86	-0.540	细胞核 Nucleus
FaLIM20	Fvb7-2:23449921..23452679	200	21 691.82	9.08	37.63	-0.376	细胞核 Nucleus
FaLIM21	Fvb7-2:23655808..23659719	430	49 337.54	8.63	48.07	-0.525	细胞核Nucleus
FaLIM22	Fvb7-2:23849991..23852934	200	21 721.91	9.08	37.48	-0.362	细胞核 Nucleus
FaLIM23	Fvb7-3:5169640..5172605	200	21 704.86	9.01	38.00	-0.353	细胞核 Nucleus
FaLIM24	Fvb7-3:6773453..6777457	506	57 325.21	8.45	52.87	-0.541	细胞核Nucleus
FaLIM25	Fvb7-3:12069028..12071004	215	23 793.94	6.50	44.19	-0.573	细胞核 Nucleus
FaLIM26	Fvb7-4:5056752..5059611	200	21 721.91	9.08	37.48	-0.362	细胞核 Nucleus
FaLIM27	Fvb7-4:5292185..5296170	506	57 277.11	8.45	51.10	-0.545	细胞核Nucleus
FaLIM28	Fvb7-4:11548941..11551124	214	23 743.92	6.54	42.18	-0.534	细胞核 Nucleus

名称 Name	染色体定位 Chromosome location	氨基酸数 Amino acids	分子量 Molecular weight	等电点 pI	不稳定指数 Instability index	亲水性 Hydrophilic	亚细胞定位 Subcellular localization
FvLIM1	Fvb1:7945940..7952121	538	60 510.36	5.20	53.78	-0.718	细胞核 Nucleus
FvLIM2	Fvb2:2982770..2998244	197	22 232.22	8.89	42.90	-0.703	细胞核 Nucleus
FvLIM3	Fvb2:5932861..5935114	527	59 323.18	5.33	52.19	-0.698	细胞核 Nucleus
FvLIM4	Fvb2:24843809..24848493	191	21 205.29	9.05	29.01	-0.539	细胞核 Nucleus
FvLIM5	Fvb5:1235177..1237221	212	23 391.44	7.52	45.56	-0.62	细胞核 Nucleus
FvLIM6	Fvb5:9381785..9385122	188	21 211.15	8.86	28.77	-0.586	细胞核 Nucleus
FvLIM7	Fvb7:12341249..12343536	217	23 962.13	6.50	45.65	-0.567	细胞核 Nucleus
FvLIM8	Fvb7:18342947..18346948	504	57 014.81	8.26	52.67	-0.541	细胞核Nucleus
FvLIM9	Fvb7:18517083..18519868	200	21 721.91	9.08	37.48	-0.362	细胞核 Nucleus
FaLIM1	Fvb1-1:20431547..20437334	534	59 915.92	5.22	52.12	-0.668	细胞核 Nucleus
FaLIM2	Fvb1-2:9164763..9171264	533	60 168.24	5.39	51.21	-0.737	细胞核 Nucleus
FaLIM3	Fvb1-3:8145568..8151824	539	60 522.40	5.20	54.39	-0.719	细胞核 Nucleus
FaLIM4	Fvb1-4:6945308..6951056	538	60 571.45	5.17	53.06	-0.712	细胞核 Nucleus
FaLIM5	Fvb2-2:728488..730564	191	21 205.29	9.05	29.01	-0.539	细胞核 Nucleus
FaLIM6	Fvb2-2:18290991..18293017	231	27 087.33	7.80	45.08	-0.512	细胞核 Nucleus
FaLIM7	Fvb2-3:2848239..2854332	491	55 136.60	5.75	44.43	-0.671	细胞核 Nucleus
FaLIM8	Fvb2-4:1747334..1750087	176	19 899.54	8.83	44.58	-0.618	细胞核 Nucleus
FaLIM9	Fvb5-1:1118664..1120389	213	23 551.67	7.92	45.67	-0.623	细胞核 Nucleus
FaLIM10	Fvb5-2:1037693..1039462	216	23 835.04	8.18	44.28	-0.569	细胞核 Nucleus
FaLIM11	Fvb5-3:18813760..18816438	188	21 172.07	8.86	31.15	-0.599	细胞核 Nucleus
FaLIM12	Fvb5-4:1027068..1028886	211	23 297.40	7.52	43.54	-0.607	细胞核 Nucleus
FaLIM13	Fvb5-4:7252377..7255038	188	21 199.10	8.86	29.68	-0.613	细胞核 Nucleus
FaLIM14	Fvb7-1:16915680..16917768	217	23 964.10	6.50	45.95	-0.579	细胞核 Nucleus
FaLIM15	Fvb7-1:25526059..25530050	506	57 362.23	8.54	52.15	-0.549	细胞核Nucleus
FaLIM16	Fvb7-1:25665993..25670535	200	21 818.02	9.08	37.58	-0.360	细胞核 Nucleus
FaLIM17	Fvb7-2:11429297..11431530	217	23 962.13	6.50	45.65	-0.567	细胞核 Nucleus
FaLIM18	Fvb7-2:17869656..17871925	217	23 949.13	6.50	45.65	-0.554	细胞核 Nucleus
FaLIM19	Fvb7-2:23292270..23296225	493	55 877.59	8.37	53.86	-0.540	细胞核 Nucleus
FaLIM20	Fvb7-2:23449921..23452679	200	21 691.82	9.08	37.63	-0.376	细胞核 Nucleus
FaLIM21	Fvb7-2:23655808..23659719	430	49 337.54	8.63	48.07	-0.525	细胞核Nucleus
FaLIM22	Fvb7-2:23849991..23852934	200	21 721.91	9.08	37.48	-0.362	细胞核 Nucleus
FaLIM23	Fvb7-3:5169640..5172605	200	21 704.86	9.01	38.00	-0.353	细胞核 Nucleus
FaLIM24	Fvb7-3:6773453..6777457	506	57 325.21	8.45	52.87	-0.541	细胞核Nucleus
FaLIM25	Fvb7-3:12069028..12071004	215	23 793.94	6.50	44.19	-0.573	细胞核 Nucleus
FaLIM26	Fvb7-4:5056752..5059611	200	21 721.91	9.08	37.48	-0.362	细胞核 Nucleus
FaLIM27	Fvb7-4:5292185..5296170	506	57 277.11	8.45	51.10	-0.545	细胞核Nucleus
FaLIM28	Fvb7-4:11548941..11551124	214	23 743.92	6.54	42.18	-0.534	细胞核 Nucleus

基序编号 Conserved motif	氨基酸数 Number of amino acids	氨基酸保守序列 Amino acid conserved sequence
motif1	50	GAVYHKSCFKCHHCKGPJKLSNYSSSEGVLYCKPHFEZLFKETGNVCKSF
motif2	50	VYPLEKVTVEGESYHKSCFRCTHGGCPJTPSNYAALEGILYCKHHFAQLF
motif3	50	AIMDTHECQPLYLEIQEFYEGLNMKVEQQVPMLLVERQALNEAMVGEKNG
motif4	50	IDMRTQPQKLTRRCEVTAILVLYGLPRLLTGSILAHEMMHAWLRLKGYPN
motif5	50	IPTNSAGLIEYRAHPFWLQKYCPSHERDRTPRCCSCERMEPKDTKYJLLD
motif6	21	FPGTRQKCKACEKTVYLVDKL
motif7	50	EKKLGEFFKHQIESDSSPAYGEGFRAGNQAVLKYGLRRTLDHIRITGSFP
motif8	21	PSKLSSMFSGTQDKCATCKKT