大白菜ACA基因家族的全基因组鉴定与表达分析

doi:10.3864/j.issn.0578-1752.2021.22.012

摘要/Abstract

摘要：

【目的】通过对大白菜ACA（Ca²⁺-ATPase）基因家族鉴定与表达分析,研究其家族基因间的共性与特性,为进一步揭示ACA家族进化关系提供数据支撑,为深入解析BraACAs在低温胁迫、盐胁迫以及自交不亲和方面的功能研究奠定基础。【方法】根据已报道的拟南芥ACA基因家族,同源比对出大白菜ACA基因家族,利用在线软件Expasy预测其分子量、理论等电点等理化性质;采用MEGA 5.0软件构建系统进化树;运用在线软件GSDS 2.0绘制基因结构图谱;TBtools对其染色体定位;McscanX软件进行拟南芥与大白菜ACA家族基因共线性分析;利用在线软件PlantCARE预测大白菜ACA基因家族启动子元件;通过在线工具Pfam和MEME进行蛋白保守结构域分析;利用qRT-PCR技术检测BraACAs在不同组织、非生物胁迫和自交异交授粉后的表达量。【结果】大白菜ACA基因家族有18个基因成员,分布在大白菜10条染色体上;根据进化树关系分成4组,分别包含3、4、4和7个成员;蛋白结构域分析显示,有13个成员包含N端自抑结构域。qRT-PCR结果表明,BraACAs主要在花与果荚中高表达;低温胁迫下,Bra002762与Bra035649表达量总体上调;盐胁迫下,Bra031701表达量显著上调;自交和异交授粉中,Bra003276和Bra024117差异性表达。对Bra002762、Bra035649、Bra031701、Bra003276和Bra024117亚细胞定位分析,发现这些基因均定位在细胞质膜上。【结论】大白菜ACA家族基因蛋白结构均含有4个ACA基因特有的高度保守结构域。该家族在大白菜不同组织中表达模式不同,5个ACA家族基因成员编码蛋白定位于细胞膜上,其中Bra002762、Bra035649、Bra031701与低温和盐胁迫响应有关;Bra003276和Bra024117与自交不亲和性相关。

关键词: ACA家族, Ca²⁺-ATPase, 低温胁迫, 盐胁迫, 自交不亲和, 大白菜

Abstract:

【Objective】The aim of this study was to identify ACA genes family from Brassica rapa, and to analyze the commonness and characteristics of those genes, which provided data support for further revealing the evolutionary relationship of ACA family. At the same time, the expression of BraACAs after self-pollination under abiotic stress was investigated, which laid the foundation for further exploring how BraACAs as regulate calcium to perform some biological functions. 【Method】Based on Arabidopsis genome database, the ACA gene family of Brassica rapa was identified by homologous alignment. The molecular weight, theoretical isoelectric point and other physicochemical properties were predicted by online software Expasy. The MEGA 5.0 software was used to construct phylogenetic tree, and the online software GSDS 2.0 was employed to draw gene structure map. According to the family gene location information, chromosome mapping was carried out in TBtools. Then, McscanX software was used to carry out the collinearity analysis of ACA family genes in Arabidopsis and Brassica rapa, and the online software PlantCARE was used to predict and analyze the promoter elements of BraACAs. The protein conserved domains were analyzed by online tools Pfam and MEME. The expression of BraACAs gene in different tissues, abiotic stress and self-pollination were detected by qRT-PCR. 【Result】A total of 18 ACA genes were systematically identified from Brassica rapa, which were distributed on 10 chromosomes. According to the phylogenetic tree, four groups were classified, including 3, 4, 4 and 7 members. According to promoter element analysis, there were many light, abiotic stress and hormone response elements in the promoter of ACA family genes in Brassica rapa, which indicated that ACA family genes had potential biological function of resisting stress. According to the analysis of protein domains, most of the ACA gene family proteins in Brassica rapa had four functional domains unique to the ACA family, 13 of which had N-terminal autoinhibitory domains. The results of qRT-PCR showed that BraACAs were mainly expressed in flowers and pods, and the expression of Bra002762 and Bra035649 genes were up-regulated under chill stress, suggesting that Bra002762 and Bra035649 had potential biological functions in response to chill stress; the expression of Bra031701 gene was up-regulated under salt stress, suggesting that Bra031701 had potential biological functions in response to salt stress. There were significant differences in the expression of Bra003276 and Bra024117 genes between self-pollination and cross-pollination, suggesting that Bra003276 and Bra024117 responded to the self-incompatibility of Brassica rapa. The subcellular localizations of these five genes were all located in the plasma membrane to verify their function of balancing ion concentration on the membrane. 【Conclusion】BraACAs protein structures contained four highly conserved domains. The expression of BraACAs was specific in tissues, and five ACA family gene members encoded proteins located on the cell membrane, of which Bra0002762, Bra035649, Bra031701 were associated with chilling and salt stress response, while Bra003276 and Bra024117 were associated with self-incompatibility reaction.

Key words: ACA gene family, Ca²⁺-ATPase, chilling stress, salt stress, self-incompatibility, Brassica rapa

王洁,吴晓宇,杨柳,段巧红,黄家保. 大白菜ACA基因家族的全基因组鉴定与表达分析[J]. 中国农业科学, 2021, 54(22): 4851-4868.

WANG Jie,WU XiaoYu,YANG Liu,DUAN QiaoHong,HUANG JiaBao. Genome-Wide Identification and Expression Analysis of ACA Gene Family in Brassica rapa[J]. Scientia Agricultura Sinica, 2021, 54(22): 4851-4868.

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基因名称 Gene name	上游引物（5´-3´） Forward primer (5´-3´)	下游引物（5´-3´） Reverse primer (5´-3´)
Bra017841	AAAAGCTTCTGGGTGTTCGTAT	GGTAGCTATCCCAACGATCAAA
Bra039940	ACCAGAATTTTGACGTTAAGGC	GAGATAAATTGGAATGCGGCTT
Bra010605	GCTGTTGGTTCAGTCGATATTC	GGCTCAACTTTCACCACTTTAC
Bra010917	GAAGCTTCAAGATGGTTCTCAC	ATTGTCTCACAAGCAGCTAGAT
Bra023790	TCGACAAGCTTACTTCTTCGAT	ATCGCTTTTCGTATTCTTTCCG
Bra033900	GTCTGCTACCACTATCTGTACC	TTGATTCTAAACCGGACCAGAA
Bra023899	TCAAGCACTAAGGAAAAACGG	ATATTTGCATACACAGAACGGC
Bra031259	AATAACGGTGATGGGTAGATCC	ATATTTGCATACACAGAACGGC
Bra016926	ATCTCTGACGTCATTGAAGGTT	AATCGCTTTAGCTGTGCTTATG
Bra003276	GCTGATGTGATCATAATGGACG	ATACAAAGTTGATGATCAGCGC
Bra007319	GTATCCATCGTGAAAAACCGAG	GTTTATATTCTGTCCGTGCACC
Bra019960	GTGATTCTGCTGATATTGGCTG	GCTATATTTGCTTGGTAGGCAC
Bra031701	TTCTGTCACGCCATTGTCCT	GGTCTATGGCAAGGCAAGGT
Bra002762	GAGAGTTCGGACATCATCATCT	GTTAATGATAAGAGCAGCGACG
Bra035649	GTAGTATTTACGTCCCAGAGGG	AGAATAGAAGACTGCGATCTGG
Bra011144	GCTCATAATACAACTGGCAGTG	GCATCGAAATTCATGCCTAACT
Bra024117	CTTGTCGTCCGCTATTTTACTG	TAGGCAAGAGTCAAGGTAACAG
Bra037404	CGGAGATAGGCAAGATACAGAG	TGTAGTTGATGATCCAGACGAG
BraActin2	CGGTGTCATGGTTGGGAGA	CGTGCTCGATGGGGTACTTC

引物名称 Primer name	正向引物（5´-3´） Forward primer(5´-3´)	反向引物（5´-3´） Reverse primer(5´-3´)
Bra002762-pCAMBIA1300-GFP	ATGACCAGTCCCTTCAAGCCA	GAGTGAACCTTCTCCAGAAGATTTTT
Bra035649-pCAMBIA1300-GFP	ATGACTAGCCTCTTCAAGCAATCTC	CAAGGCTTCAAAAGTTTGTTGTTT
Bra031701-pCAMBIA1300-GFP	ATGGAAGACGCTTACGCCAG	CGCCGGTCCTTGGGGAGT
Bra024117-pCAMBIA1300-GFP	ATGAGTGGTGGACAAGGACAGTT	ACCTGATGAATTCCTTCTCCATCG
Bra003276-pCAMBIA1300-GFP	ATGTCTAATCTCCTCAAGGATTTTCA	GGCAGAGTCAGATGGACCAGAA

基因ID Gene ID	理论等电点 pI	分子量 Molecular weight (Da)	CDS长度 CDS length (bp)	氨基酸 No. of amino acids (aa)	亚细胞定位 Subcellular location	跨膜结构数量 Number of membrane spanning domain	拟南芥对应ID Arabidopsis corresponds ID
Bra017841	5.58	110370.20	3045	1015	细胞质膜Plas	8	AT4G37640
Bra039940	5.65	110609.40	3045	1015	细胞质膜Plas	8	AT2G22950
Bra010605	5.45	112318.20	3093	1031	细胞质膜Plas	8	AT4G37640
Bra010917	5.40	110689.40	3054	1017	细胞质膜Plas	8	AT1G27770
Bra023790	8.75	112419.20	3054	1018	细胞质膜Plas	8	AT3G22910
Bra033900	8.28	112171.60	3048	1016	细胞质膜Plas	8	AT3G22910
Bra023899	6.16	119249.50	3291	1097	细胞质膜Plas	10	AT3G21180
Bra031259	6.09	119273.50	3288	1096	细胞质膜Plas	8	AT3G21180
Bra016926	5.43	112256.90	3099	1033	细胞质膜Plas	8	AT2G41560
Bra003276	6.08	111847.50	3078	1026	细胞质膜Plas	8	AT3G57330
Bra007319	5.84	111456.90	3066	1022	细胞质膜Plas	8	AT3G57330
Bra019960	5.06	113187.20	3138	1046	细胞质膜Plas	7	AT1G10130
Bra031701	5.26	118294.80	3256	1086	细胞质膜Plas	8	AT1G10130
Bra002762	8.03	116025.10	3222	1074	细胞质膜Plas	7	AT5G57110
Bra035649	7.81	116661.70	3231	1077	细胞质膜Plas	7	AT5G57110
Bra011144	6.48	116775.40	3210	1070	细胞质膜Plas	8	AT5G57110
Bra024117	6.39	115856.20	3186	1062	细胞质膜Plas	8	AT4G29900
Bra037404	5.31	115615.30	3165	1055	细胞质膜Plas	8	AT4G00900
平均数 Average	6.28	114171.00	3142	1047	\	\	\