辣椒GASA基因家族的鉴定及分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0878

摘要/Abstract

摘要：

目的研究辣椒（Capsicum annuum L.）GASA的基因结构和表达分析，为阐明CaGASA基因的生物学功能及进一步培育抗逆性强的辣椒品种提供参考。方法运用生物信息学方法对辣椒8个GASA进行鉴定，并分析它们的理化性质、系统发育关系、基因结构、保守基序、染色体位置、蛋白互作网络、基因同源性和启动子区域等，实时荧光定量PCR（real-time fluorescence quantitative PCR, RT-qPCR）验证辣椒GASA基因家族成员在不同组织、非生物胁迫和激素处理下的表达模式。结果 8个CaGASA在辣椒5条染色体上不均匀分布，所有CaGASA蛋白都有一个共同的结构域GASA；亚细胞定位预测结果表明，CaGASA蛋白存在于细胞外区域；辣椒、拟南芥（Arabidopsis thaliana）、番茄（Solanum lycopersicum L.）、水稻（Oryza sativa L.）基因组的直系同源GASA基因共线性分析结果表明，辣椒与其他3个物种之间有14个共线性事件，CaGASA5与3个物种的GASA基因均具有共线性；顺式元件分析表明，GASA基因可能受到多种植物激素和胁迫的诱导；转录组数据分析显示，CaGASA在辣椒花和果实中的表达水平高于叶；RT-qPCR分析表明，CaGASA基因响应低温、高温胁迫和脱落酸（abscisic acid, ABA）、赤霉素（gibberellin, GA）、吲哚乙酸（indole acetic acid, IAA）和茉莉酸（jasmonic acid, MeJA）激素处理。结论辣椒GASA基因家族可能参与辣椒花发育，响应激素和耐高、低温胁迫的应答。

关键词: 辣椒, GASA基因家族, 基因家族鉴定, 生物信息学分析, 表达分析

Abstract:

Objective The gene structure and expression analysis of GASA in pepper (Capsicum annuum L.) were studied to provide reference for elucidating the biological function of CaGASA gene and further cultivating pepper varieties with strong stress resistance. Method Bioinformatics method was applied to identify eight GASA family members in pepper, and systematically analyze their physical and chemical properties, phylogenetic relationships, gene structures, conserved motifs, chromosomal locations, gene synteny analyses, and promoter regions. Real-time fluorescence quantitative PCR (RT-qPCR) was used to verify the expression patterns of GASA gene family members in the pepper under different tissues, abiotic stresses and hormone treatments. Result Eight CaGASA proteins were distributed unevenly on the five chromosomes of pepper, and all CaGASA proteins had a common domain. The prediction of subcellular localization indicated that CaGASA protein existed in the extracellular region. The results of co-linear analysis of GASA gene in pepper, Arabidopsis, tomato (Solanum lycopersicum L.) and rice (Oryza sativa L.) showed that there were 14 co-linear events between pepper and other three species. CaCAGA5 was found to be co-linear with that of the three species. According to the cis-elements analyses, CaGASA genes may be induced by a variety of phytohormones and stresses. Transcriptome data analysis showed that the expression of CaGASA in the flower and fruit was higher than that in the leaf. RT-qPCR analysis showed that CaGASA gene was induced by low and high temperature stress and abscisic acid, gibberellin, indole acetic acid and jasmonic acid hormones. Conclusion The GASA gene family may be involved in pepper flower development, response to hormones and response to high and low temperature stress.

Key words: pepper, GASA gene family, identification of gene family, bioinformatic analysis, expression analysis

王田田, 常雪瑞, 黄婉洋, 黄嘉欣, 苗如意, 梁燕平, 王静. 辣椒GASA基因家族的鉴定及分析[J]. 生物技术通报, 2025, 41(4): 166-175.

WANG Tian-tian, CHANG Xue-rui, HUANG Wan-yang, HUANG Jia-xin, MIAO Ru-yi, LIANG Yan-ping, WANG Jing. Identification and Analysis of GASA Gene Family in Pepper （Capsicum annuum L.）[J]. Biotechnology Bulletin, 2025, 41(4): 166-175.

图/表 11

表1 CaGASA家族基因表达分析的RT-qPCR引物

Table 1 RT-qPCR primers for expression analysis of CaGASA family genes

基因 Gene	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）
CaGASA1	CATTCCAAAAGGCATTTGCT	GCAAGGGCATGCTTCATAAT
CaGASA2	TGCAGCTTTGCTTATTGCAT	GTACGCAGTGGCATCTGTTG
CaGASA3	GCAGCAGCAACAACAAAGAA	TAGCAAGGGCAAGTTTGCTT
CaGASA4	TGATGCCCAGTACCACATTG	GGTCCTCCTTCCTTGGTCTT
CaGASA5	ACTTGAACAACGGGGATTTG	AAGGGCACTTGGTCTTGTTG
CaGASA6	CCACTGCACCGACATACATT	TGGTGGAACACATTGACACC
CaGASA7	TCTCTCCTTGCTCCTTCTCG	GCAAGGGCAAGTCTGAGTGT
CaGASA8	TGAGCTTGTGGAAGCTGATG	CACCATGGGTAAGCATGTCA
β-Actin	CAACATCTTCACTCTCTGCTCT	ACTAGGAAAAACAGCACTTGGT

表2 辣椒GASA基因家族成员信息

Table 2 Information of GASA gene family members in pepper

基因号 Gene ID	基因 Gene	氨基酸数 Number of amino acids/aa	分子量 Molecular weight/kD	等电点 Theoretical pI	不稳定指数 Instability index	脂肪指数 Aliphatic index	总平均亲水系数 Grand average of hydropathicity	亚细胞定位 Subcellular localization	跨膜结构域 Transmembrane domain
Capana02g002587	CaGASA1	102	11.15	9.01	46.06	49.90	-0.288	细胞外	0
Capana02g002588	CaGASA2	101	10.97	9.12	72.87	56.14	-0.166	细胞外	1
Capana02g003203	CaGASA3	112	12.67	9.42	42.06	53.04	-0.478	细胞外	0
Capana03g000783	CaGASA4	104	11.46	9.35	45.52	62.88	-0.026	细胞外	1
Capana03g001010	CaGASA5	145	15.74	9.27	39.20	81.38	-0.043	细胞外	0
Capana06g002956	CaGASA6	113	12.55	9.46	48.91	73.27	0.103	细胞外	1
Capana08g002677	CaGASA7	104	11.18	9.24	37.42	71.44	-0.138	细胞外	0
Capana12g000591	CaGASA8	113	12.27	7.97	36.80	79.38	0.040	细胞外	0

图1 辣椒GASA蛋白的三级结构预测

Fig. 1 Predicted three-dimensional structures of GASA proteins in pepper

图2 CaGASA基因在染色体上的位置

Fig. 2 Chromosome location of CaGASA family genes

图3 CaGASA蛋白互作网络分析图T459_27842：含l9抑制子蛋白域蛋白；T459_18858：假定的嘌呤通透酶5；T459_18848：假定的嘌呤通透酶5；T459_03219：含Fe2OG双加氧酶结构域蛋白；T459_17925、T459_24899、T459_28071、T459_18373、T459_16468、T459_05110：未鉴定的蛋白。紫线表示实验测定；蓝线表示共同产生；黄线表示文本挖掘证据；黑线表示共表达

Fig. 3 Interaction network analysis of CaGASA proteinsT459_27842:Inhibitor I9 domain-containing protein. T459_18858: Putative purine permease 5. T459_18848: Putative purine permease 5. T459_03219: Fe2OG dioxygenase domain-containing protein. T459_17925, T459_24899, T459_28071, T459_18373, T459_16468, T459_05110: Uncharacterized protein. Purple line indicates experimental determination; the blue line indicates co-occurrence; the yellow line indicates text mining evidence; and the black line indicates co-expression

图4 辣椒（Ca）和拟南芥（At）、番茄（Sl）、水稻（Os）GASA家族成员系统发育树

Fig. 4 Phylogenetic tree of GASA family members of Arabidopsis thaliana (At), Solanum lycopersicum (Sl), Oryza sativa (Os), and Capsicum annuum (Ca)

图5 辣椒GASA家族蛋白结构预测

Fig. 5 Prediction of GASA family protein structure in pepper

图6 辣椒（Ca）和拟南芥（At）、番茄（Sl）、水稻（Os）GASA基因的同源分析

Fig. 6 Synteny analysis of GASA genes among C. annuum (Ca), A. thaliana (At), S. lycopersicum (Sl), and O. sativa (Os)

图7 CaGASA家族启动子顺式元件预测

Fig. 7 Cis-acting element prediction in the CaGASA promoters

图8 辣椒GASA家族基因在不同组织中的表达分析L1‒L9代表新叶刚出现后2、5、10、15、20、30、40、50和60 d；F1‒F9代表出现花蕾后；G1‒G11代表授粉后10、15、20、25、30、35、40、45、50、55和60 d；ST1、ST2代表授粉后10和15 d；S3‒S11代表授粉后20、25、30、35、40、45、50、55和60 d；T3‒T11代表授粉后20、25、30、35、40、45、50、55和60 d

Fig. 8 Expression analysis of GASA in different tissues in pepperL1‒L9 refers to 2, 5, 10, 15, 20, 30, 40, 50, and 60 d after new leaf emergence; F1‒F9 refers to 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 d after flower bud emergence; G1‒G11 refers to 10, 15, 20, 25 30, 35, 40, 45, 50, 55, and 60 d after pollination; St1 and St2 refers to 10, 15 d after pollination; S3‒S11 refer to 20, 25, 30, 35, 40, 45, 50, 55, and 60 d after pollination; T3‒T11 refers to 20, 25, 30, 35, 40, 45, 50, 55, and 60 d after pollination

图9 八个CaGASA基因在非生物胁迫处理下的相对表达量*、**、***分别表示在0.05、0.01、0.001水平上差异显著

Fig. 9 Relative expressions of eight CaGASA genes under abiotic stress treatment*, **, and *** indicate significant differences at the level of 0.05, 0.01 and 0.001 respectively

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