苹果LIM基因家族生物信息学及表达分析

doi:10.3864/j.issn.0578-1752.2019.23.013

摘要/Abstract

摘要：

【目的】在苹果全基因组中鉴定LIM,通过分析启动子作用元件、保守结构域、基因聚类、基因结构、染色体定位以及组织表达模式,为研究和利用苹果LIM奠定基础。【方法】利用苹果基因组数据库GDR和PLAZA,获得苹果LIM家族成员并进行编号。苹果LIM蛋白氨基酸序列的基本信息通过ExPASy Proteomics Server进行预测,利用Cell-PLoc进行亚细胞定位预测,利用CD-Search Tool进行LIM结构域分析,采用MEGA 7软件构建进化树,采用GSDS绘制基因结构,并利用TBtools软件对鉴定得到的MdLIM进行染色体定位,通过实时荧光定量RT-PCR对MdLIM的组织表达进行分析,并利用SPSS 18.0软件分析差异显著性。【结果】共鉴定得到11个苹果LIM家族成员,这些MdLIM蛋白包含96—222个不等的氨基酸残基,等电点分布在6.14—9.01。亚细胞定位结果显示,MdLIM蛋白在细胞核中均有分布。启动子作用元件分析表明,11个MdLIM启动子上分布有响应激素、环境适应性和逆境诱导的元件。蛋白保守结构域分析表明,11个MdLIM蛋白中除MdLIM8具有单LIM结构域外,其余10个均具有双LIM结构域。根据聚类分析结果可将MdLIM分为4组。染色体定位结果显示,MdLIM分布在苹果17条染色体中的7条,且MdLIM在7条染色体上的分布不均匀。花、叶、果皮和茎中的实时荧光定量RT-PCR结果显示,4个组织中均能检测到MdLIM的表达,且表达量具有一定差异。【结论】苹果LIM基因家族包括11个成员,进化上可分为4组,11个基因分布于7条染色体上,在不同组织中的表达具有多样性和特异性。

关键词: 苹果, 全基因组, LIM家族, 生物信息学, 表达分析

Abstract:

【Objective】 In order to lay a basis for the further functional research and application of MdLIM genes, this study were carried out to analyze the bioinformatics (e.g promoter action element, conserved domain, gene clustering, gene structure, chromosome localization) and expression of the LIM gene family in apple. 【Method】Based on the apple genome database GDR and PLAZA, the members of LIM gene were identified. The MdLIM amino acid sequence prediction, subcellular localization prediction, LIM domain analysis, and phylogenetic tree the gene structure were completed by ExPASy Proteomics Server, Cell-PLoc, CD-Search Tool, MEGA7, and GSDS, respectively. In addition, the expression pattern of MdLIM genes in different tissues and in peels with different degree of fruit russeting in samples was analyzed by real-time qRT-PCR.【Result】A total of eleven MdLIM genes were identified from apple genome. These MdLIM proteins contained 96-222 amino acid residues with isoelectric points ranging from 6.14 to 9.01. The results of subcellular localization showed that the apple LIM proteins were distributed in the nucleus. Analysis of promoter showed these 11 MdLIM genes contained cis-acting elements related to hormone responses, environmental adaptability and adversity induction. Conserved domains showed that ten MdLIM proteins had double LIM domains except MdLIM8. According to the phylogeny relationship, MdLIM genes were divided into four categories. The expression patterns of the 11 MdLIM genes in flowers, leaves, fruit peels and stems were determined by real-time RT-PCR, and the results showed that their diverse and specific expression could be detected in all of the four tissues, suggesting that they might play different roles in different tissues. 【Conclusion】Eleven MdLIM genes were identified from the whole genome of apple, and they could be divided into four groups, and distributed on 7 chromosomes with diverse and specific tissues expression patterns.

Key words: apple, genome-wide, LIM family, bioinformatics, expression analysis

袁高鹏, 韩晓蕾, 卞书迅, 张利义, 田义, 张彩霞, 丛佩华. 苹果LIM基因家族生物信息学及表达分析[J]. 中国农业科学, 2019, 52(23): 4322-4332.

YUAN GaoPeng, HAN XiaoLei, BIAN ShuXun, ZHANG LiYi, TIAN Yi, ZHANG CaiXia, CONG PeiHua. Bioinformatics and Expression Analysis of the LIM Gene Family in Apple[J]. Scientia Agricultura Sinica, 2019, 52(23): 4322-4332.

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基因 Gene	上游引物Forward primer (5′-3′)	下游引物Reverse primer (5′-3′)
MdLIM1	ATGCAGCGCCTGTGATAAGA	GCTCGAAATGAGGTTTGCAGT
MdLIM2	GCTACTTGTGGTAAAACCGCT	CCAAAGCCGCGTAGTTTGAG
MdLIM3	ATGCCAAGTCAGTCTCAAACT	TGGTATGGAGTCCCGTTG
MdLIM4	GGGACCGTAGACAAATGTGC	AGTTGGGCAAAGTGGTGCTT
MdLIM5	AAAACTGTTCGAGAAAGATCCG	GAAGAGTTGGCTATGGTGATGC
MdLIM6	GACAGTCTATCCGCTGGAGA	ACGATGAATGTGTAAGGGCA
MdLIM7	GATGGGATTTCCTACCACAA	CAGTCTCCTTGAACAGTTGCT
MdLIM8	TCTCCTTTCTCAGGGACACC	GCACTTATCTCTGGTTCCACC
MdLIM9	TGCCTACCATAAGACCTGCT	TCTCCTTGAAGAGTTGCTCG
MdLIM10	ACTAGTTCCAGTACACAGACCAAC	CTCCATGAGTGCACCGGAAA
MdLIM11	TCTACCACAAAGCCTGCTTCC	CAAGACTGCCGGTTCTTTTGA
MdActin	TGACCGAATGAGCAAGGAAATTACT	TACTCAGCTTTGGCAATCCACATC

基因 Gene	基因登录号 Gene accession No.	染色体定位 Chromosome location	大小 Size (aa)	分子量 Molecular weight (D)	等电点 Isoelectric point	亚细胞定位 Subcellular Localization
MdLIM1	MD01G1054500	Chr01:15895562..15897359	219	23939.82	6.14	细胞核Nucleus
MdLIM2	MD01G1145700	Chr01:25554814..25556454	199	21649.78	9.01	细胞核Nucleus
						高尔基体Golgi apparatus
MdLIM3	MD05G1101500	Chr05:20964629..20966107	197	22242.34	8.73	细胞核Nucleus
MdLIM4	MD06G1078000	Chr06:19291196..19292843	213	23595.59	7.51	细胞核Nucleus
MdLIM5	MD06G1178800	Chr06:31881986..31883872	188	21297.27	8.86	细胞核Nucleus
MdLIM6	MD07G1140900	Chr07:20424546..20426430	221	24195.19	6.31	细胞核Nucleus
MdLIM7	MD07G1212800	Chr07:29070702..29074084	200	21720.86	9.01	细胞核Nucleus
MdLIM8	MD08G1081300	Chr08:6745950..6747349	96	10755.12	7.03	细胞核Nucleus
MdLIM9	MD14G1098800	Chr14:15093296..15094757	222	24598.9	8.49	细胞核Nucleus
MdLIM10	MD14G1185200	Chr14:27756572..27758485	188	21254.15	8.77	细胞核Nucleus
MdLIM11	MD15G1068200	Chr15:4725341..4726746	195	21931.05	8.87	细胞核Nucleus
						高尔基体Golgi apparatus