西洋参bZIP基因家族全基因组鉴定和表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0970

摘要/Abstract

摘要：

【目的】碱性亮氨酸拉链转录因子（the basic leucine zipper，bZIP）是植物中最大的转录因子家族之一，参与植物生长发育及激素调控等多个进程，对西洋参bZIP基因家族进行鉴定并对其结构及表达模式进行分析，为深入研究西洋参bZIP基因家族功能提供理论依据。【方法】利用生物信息学对西洋参bZIP基因进行理化性质、系统发育关系、基因结构、保守基序、顺式作用元件、染色体定位、进化选择压力分析、以及组织特异性表达分析，同时利用外源激素ABA处理西洋参后进行转录组数据分析及荧光定量验证。【结果】西洋参中共鉴定出121个bZIPs基因；根据系统发育树，这些基因被分为12个亚组；基因结构和保守基序分析表明，同一亚组的蛋白具有相似的保守基序和结构域，保守基序1在西洋参bZIP蛋白质中分布最广，保守性最强；顺式作用元件分析表明，bZIP上游启动子元件存在激素响应元件、防御和应激反应元件，其中ABA激素响应元件占比最大；染色体定位分析显示bZIP基因不均匀的分布在24条染色体上；进行进化选择压力分析，发现纯化选择在西洋参bZIP基因家族进化过程中起着重要作用；组织特异性表达分析，PqbZIPs基因在花和根中的表达量相对较高；转录组数据发现bZIP转录因子积极响应ABA激素处理，随后进行荧光定量实验验证，发现基因表达变化趋势基本一致。【结论】PqbZIP基因家族成员具有组织特异性表达模式且多数成员响应ABA激素处理。

关键词: bZIP, 转录因子, 西洋参, ABA, 转录组

Abstract:

【Objective】The basic leucine zipper (bZIP) is one of the largest transcription factor families in plants, involved in multiple processes such as plant growth and hormone regulation. This study aims to identify the bZIP gene family in Panax quinquefolius and analyze its structure and expression patterns, providing a theoretical basis for further research on the function of the bZIP gene family in P. quinquefolius.【Method】Bioinformatics was used to analyze the physicochemical properties, phylogenetic relationships, gene structure, conserved motifs, cis acting elements, chromosomal localization, evolutionary selection pressure,and tissue-specific expression of the bZIP gene in P. quinquefolius. Simultaneously, exogenous hormone ABA was used to treat P. quinquefolius for transcriptome data analysis and RT-qPCR.【Result】A total of 121 bZIPs genes were identified in P. quinquefolius. According to the phylogenetic tree, these genes were divided into 12 subgroups. Gene structure and conserved motif analysis showed that proteins in the same subgroup had similar conserved motifs and domains, with conserved motif 1 being the most widely distributed and highly conserved in the bZIP protein of P. quinquefolius. The analysis of cis-acting elements showed that there were hormone responsive elements, defense and stress responsive elements in the upstream promoter element of bZIP, with ABA hormone responsive elements accounting for the largest proportion. Chromosome localization analysis showed that bZIP genes were unevenly distributed on 24 chromosomes. Conducting evolutionary selection pressure analysis, it was found that purification selection played an important role in the evolution of the bZIP gene family in P. quinquefolius; organizational specific expression analysis showed that the expression levels of PqbZIPs genes were relatively high in flowers and roots; transcriptome data revealed that bZIP transcription factors actively responded to ABA hormone treatment. Subsequently, RT-qPCR experiments were conducted to verify that the trend of gene expression changes was basically consistent. 【Conclusion】Members of the PqbZIP gene family have tissue-specific expression patterns and most respond to ABA hormone treatment.

Key words: bZIP, transcription factors, Panax quinquefolius, ABA, transcriptome

郭纯, 宋桂梅, 闫艳, 邸鹏, 王英平. 西洋参bZIP基因家族全基因组鉴定和表达分析[J]. 生物技术通报, 2024, 40(4): 167-178.

GUO Chun, SONG Gui-mei, YAN Yan, DI Peng, WANG Ying-ping. Genome Wide Identification and Expression Analysis of the bZIP Gene Family in Panax quinquefolius[J]. Biotechnology Bulletin, 2024, 40(4): 167-178.

图/表 11

表1 PqbZIP基因家族实时荧光定量引物

Table 1 Real-time fluorescence quantitative primers of PqbZIP gene family

基因 Gene	上游引物Forward primer（5'-3'）	下游引物Reverse primer（5'-3'）
PqbZIP1778	TGATACATCTTCTGTGTCACCAATTCC	TGCCTCCTCTCAACAACCTTCTC
PqbZIP3076	GCCAGAGCCAGCAACAAGTTC	GATCTCTTCATCACTCTCCATACCTTC
PqbZIP53695	GTGCCACAACTCAACCAGAAGG	TCCGACTCCATCCAAGGTGTTC
PqbZIP53753	TGATGAACGATAACGGCATTGATAGG	TCCAGTAGAATATCCGCCAAGAGG
GAPDH1-F	GCTATCAAGGAGGAGTCGGAGGG	CTGGACAGATGCCATGTGAACAA

图1 PqbZIPs蛋白的系统发育树不同的颜色表示不同的亚科

Fig. 1 Phylogenetic tree of PqbZIPs proteins Different colors representing different subfamilies

图2 PqbZIP家族中的基因结构示意图和保留的基序模式（A）及顺式作用元件分析（B）

Fig. 2 Schematic diagram of gene structure and retained motif patterns in the PqbZIP family (A) and Cis-acting element analysis (B)

图3 PqbZIP家族保守结构域的可视化每个位置的字母堆的总高度表示该位置的序列守恒。字母堆中单个字母的高度代表该位置对应氨基酸的相对频率

Fig. 3 Visualization of conservative domains in the PqbZIP family The total height of the letter stack at each position indicates the conservation of the sequence at that position. The height of a single letter in the letter stack indicates the relative frequency of the corresponding amino acid at that position

图4 西洋参bZIP基因家族的染色体定位及共线性分析绿线表示重复的西洋参bZIP基因对

Fig. 4 Chromosomal localization and collinearity analysis of the bZIP gene family in P. quinquefolius The green line indicates duplicate pairs of bZIP genes in P. quinquefolius

表2 PqbZIPs大规模复制事件的复制模式

Table 2 Replication mode of PqbZIPs for large-scale replication events

Sequence	Method	Ka	Ks	Ka/Ks	Length/bp	Sequence	Method	Ka	Ks	Ka/Ks	Length/bp
bZIP2-bZIP43	MA	0.117946	0.165607	0.712204	960	bZIP67-bZIP1	MA	0.097071	0.323816	0.299772	1 152
bZIP3-bZIP44	MA	0.0130427	0.0128818	1.01249	489	bZIP70-bZIP19	MA	0.0142327	0.0409637	0.347448	1 332
bZIP10-bZIP38	MA	0.100549	0.140421	0.716056	846	bZIP73-bZIP14	MA	0.0547414	0.0672358	0.81417	702
bZIP10-bZIP112	MA	0.11451	0.521399	0.219622	879	bZIP76-bZIP36	MA	0.110057	0.324559	0.339097	564
bZIP11-bZIP37	MA	0.0942089	0.0981423	0.959922	957	bZIP79-bZIP104	MA	0.0904191	0.339624	0.266233	462
bZIP13-bZIP76	MA	0.104919	0.30191	0.347518	564	bZIP87-bZIP81	MA	0.317343	0.931453	0.340696	1 065
bZIP13-bZIP109	MA	0.101853	0.290082	0.351118	564	bZIP92-bZIP51	MA	0.015582	0.0313318	0.497324	903
bZIP15-bZIP54	MA	0.105643	0.321516	0.328579	972	bZIP98-bZIP52	MA	0.0483138	0.0459249	1.05202	1 023
bZIP15-bZIP55	MA	0.156553	0.426652	0.366934	1 197	bZIP100-bZIP80	MA	0.0367017	0.113648	0.322942	1 086
bZIP16-bZIP80	MA	0.0682639	0.322732	0.211519	1 128	bZIP101-bZIP83	MA	0.0497949	0.0973565	0.51147	1 368
bZIP16-bZIP100	MA	0.0627594	0.322994	0.194305	1 083	bZIP105-bZIP60	MA	0.0758902	0.29116	0.260647	1 218
bZIP36-bZIP109	MA	0.106955	0.312518	0.342235	564	bZIP108-bZIP53	MA	0.0113524	0.0549189	0.206712	1 002
bZIP45-bZIP20	MA	0.0126196	0.0519384	0.242973	435	bZIP114-bZIP26	MA	0.0106575	0.0442045	0.241095	846
bZIP46-bZIP24	MA	0.0182878	0.0429762	0.425534	1 533	bZIP115-bZIP81	MA	0.242357	0.752788	0.321946	1 068
bZIP47-bZIP23	MA	0.018861	0.0298436	0.631996	993	bZIP115-bZIP82	MA	0.16807	0.619685	0.271218	1 308
bZIP48-bZIP21	MA	0.0890243	0.194441	0.457848	663	bZIP118-bZIP85	MA	0.114442	0.317447	0.360506	894
bZIP50-bZIP95	MA	0.119225	0.411079	0.29003	843	bZIP118-bZIP103	MA	0.0895535	0.261122	0.342957	885
bZIP61-bZIP91	MA	0.168025	0.327658	0.512807	771	bZIP119-bZIP32	MA	0.0231439	0.0836963	0.276523	996

表2 PqbZIPs大规模复制事件的复制模式

Table 2 Replication mode of PqbZIPs for large-scale replication events

Sequence	Method	Ka	Ks	Ka/Ks	Length/bp	Sequence	Method	Ka	Ks	Ka/Ks	Length/bp
bZIP2-bZIP43	MA	0.117946	0.165607	0.712204	960	bZIP67-bZIP1	MA	0.097071	0.323816	0.299772	1 152
bZIP3-bZIP44	MA	0.0130427	0.0128818	1.01249	489	bZIP70-bZIP19	MA	0.0142327	0.0409637	0.347448	1 332
bZIP10-bZIP38	MA	0.100549	0.140421	0.716056	846	bZIP73-bZIP14	MA	0.0547414	0.0672358	0.81417	702
bZIP10-bZIP112	MA	0.11451	0.521399	0.219622	879	bZIP76-bZIP36	MA	0.110057	0.324559	0.339097	564
bZIP11-bZIP37	MA	0.0942089	0.0981423	0.959922	957	bZIP79-bZIP104	MA	0.0904191	0.339624	0.266233	462
bZIP13-bZIP76	MA	0.104919	0.30191	0.347518	564	bZIP87-bZIP81	MA	0.317343	0.931453	0.340696	1 065
bZIP13-bZIP109	MA	0.101853	0.290082	0.351118	564	bZIP92-bZIP51	MA	0.015582	0.0313318	0.497324	903
bZIP15-bZIP54	MA	0.105643	0.321516	0.328579	972	bZIP98-bZIP52	MA	0.0483138	0.0459249	1.05202	1 023
bZIP15-bZIP55	MA	0.156553	0.426652	0.366934	1 197	bZIP100-bZIP80	MA	0.0367017	0.113648	0.322942	1 086
bZIP16-bZIP80	MA	0.0682639	0.322732	0.211519	1 128	bZIP101-bZIP83	MA	0.0497949	0.0973565	0.51147	1 368
bZIP16-bZIP100	MA	0.0627594	0.322994	0.194305	1 083	bZIP105-bZIP60	MA	0.0758902	0.29116	0.260647	1 218
bZIP36-bZIP109	MA	0.106955	0.312518	0.342235	564	bZIP108-bZIP53	MA	0.0113524	0.0549189	0.206712	1 002
bZIP45-bZIP20	MA	0.0126196	0.0519384	0.242973	435	bZIP114-bZIP26	MA	0.0106575	0.0442045	0.241095	846
bZIP46-bZIP24	MA	0.0182878	0.0429762	0.425534	1 533	bZIP115-bZIP81	MA	0.242357	0.752788	0.321946	1 068
bZIP47-bZIP23	MA	0.018861	0.0298436	0.631996	993	bZIP115-bZIP82	MA	0.16807	0.619685	0.271218	1 308
bZIP48-bZIP21	MA	0.0890243	0.194441	0.457848	663	bZIP118-bZIP85	MA	0.114442	0.317447	0.360506	894
bZIP50-bZIP95	MA	0.119225	0.411079	0.29003	843	bZIP118-bZIP103	MA	0.0895535	0.261122	0.342957	885
bZIP61-bZIP91	MA	0.168025	0.327658	0.512807	771	bZIP119-bZIP32	MA	0.0231439	0.0836963	0.276523	996

图5 西洋参与人参、西洋参与三七之间的bZIP同源性分析背景上的灰色线条表示西洋参和其他植物基因组中的共线块；绿线突出相同基因的西洋参bZIP基因对

Fig. 5 Homology analysis of bZIPs between P. quinquefoli-us and Panax ginseng as well as P. quinquefolius and Panax notoginseng The gray lines on the background indicate collinear blocks in the genomes of P. quinquefolius and other plants; the green line highlights the bZIP gene pair of homologous P. quinquefolius

图6 PqbZIPs基因在根、叶、花组织表达模式分析

Fig. 6 Expression pattern analysis of PqbZIPs gene in the root, leaf, and flower tissues

图7 PqbZIPs基因差异表达的韦恩图

Fig. 7 Venn diagram of differential expression of the Pqb-ZIP gene

图8 PqbZIPs基因在ABA胁迫下的表达模式分析

Fig. 8 Expression pattern analysis of PqbZIPs genes under ABA stress

图9 在ABA处理下4个PqbZIPs基因的相对表达量不同小写字母表示P < 0.05时差异显著

Fig. 9 Relative expressions of four PqbZIPs genes under ABA treatment Different lowercase letters indicate significant differences when P<0.05

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