基于冬凌草全长转录组的<i>TIFY</i>基因家族鉴定与表达分析

练从龙; 兰金旭; 杨晶凡; 李晶晶; 杨灏; 陈随清

doi:10.19303/j.issn.1008-0384.2024.03.006

基于冬凌草全长转录组的TIFY基因家族鉴定与表达分析

doi: 10.19303/j.issn.1008-0384.2024.03.006

河南中医药大学药学院 / 河南省中药资源与中药化学重点实验室 / 豫药全产业链研发河南省协同创新中心，河南　郑州　450046

基金项目: 河南省科技研发计划联合基金项目（优势学科培育类）（232301420078）；河南省中药材产业技术体系项目（豫科项〔2022〕30号）；中医药公共卫生服务补助专项（豫财社〔2019〕40号）

详细信息

作者简介:
练从龙（1988 — ），男，博士，讲师，主要从事药用植物生理生态与分子生物学研究，E-mail：770379183@qq.com

通讯作者:
陈随清（1965 — ），男，博士，教授，主要从事中药资源与质量评价研究，E-mail：chsq@hactcm.edu.cn

中图分类号: R286
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出版历程
- 收稿日期: 2023-10-11
- 修回日期: 2023-12-02
- 网络出版日期: 2024-03-28
- 刊出日期: 2024-03-28

Identification and Expressions of TIFY Family Based on the Full-Length Transcriptome in Isodon rubescens

School of Pharmacy, Henan University of Chinese Medicine / Henan Key Laboratory of Chinese Medicine Resources and Chemistry / Collaborative Innovation Center of Research and Development on the Whole Industry Chain of Yu-Yao, Zhengzhou, Henan　450046, China

摘要

摘要: 目的 TIFY 蛋白是茉莉酸（JA）信号通路的关键调节因子，在植物生长发育、非生物胁迫以及次生代谢产物的积累中具有显著的调控作用，揭示冬凌草（Isodon rubescens）的TIFY基因可为冬凌草的抗逆性改良育种和次生代谢产物合成研究提供理论依据。方法基于冬凌草三代全长转录组序列，通过生物信息学方法对冬凌草TIFY基因家族进行鉴定和分析，并采用RT-qPCR 技术分析其在不同组织中的表达特性。结果（1）成功从冬凌草中鉴定出12个TIFY家族基因成员；（2）理化性质分析表明，其氨基酸长度124～378 aa，分子量13 924.89～39 692.38 Da，等电点5.05～9.69；除IrTIFY10蛋白为稳定蛋白，其他均为不稳定蛋白；IrTIFYs蛋白的亚细胞定位均在细胞核，均为亲水性蛋白，且不含信号肽。（3）结构分析表明，IrTIFY家族蛋白成员无跨膜结构，二级结构中含量最多的结构类型为无规则卷曲；且含有多个磷酸化位点。（4）密码子偏好性分析结果表明，IrTIFY基因家族密码子偏好性较弱，稍倾向于使用以A或U结尾的密码子。（5）启动子元件分析表明冬凌草TIFY家族成员中存在多个光响应元件、激素响应元件和逆境响应元件等，但不同成员之间的元件存在差异。（6）进化树分析表明，冬凌草TIFY家族12个成员分为PPD（IrTIFY2）、ZML（IrTIFY3/8/10）、TIFY（IrTIFY7/12）和JAZ（IrTIFY1/4/5/6/9/11）4个亚家族，进化上与同为唇形科的丹参（Salvia miltiorrhiza）亲缘关系最近。（7）RT-qPCR分析结果显示冬凌草TIFY家族12个成员在不同组织中的表达量均表现为叶＞茎＞根，且大部分成员存在显著差异。结论 TIFY基因家族在冬凌草的生长发育过程中可能发挥重要的调控作用，并且可能参与调控冬凌草次生代谢产物的合成，为进一步深入研究冬凌草TIFY基因家族的功能奠定基础和提供了思路。
- 冬凌草 /
- TIFY基因家族 /
- 生物信息学分析 /
- 表达分析
Abstract: Objective TIFY protein is a key regulator of the JA signalling pathway and plays a significant regulatory role in plant growth and development, abiotic stress and the accumulation of secondary metabolites. The identification of the TIFY gene in Isodon rubescens provides a theoretical foundation for the breeding of I. rubescens with enhanced stress tolerance and the investigation of the synthesis of secondary metabolites. Method TIFY family was identified using bioinformatic methods based on the full-length transcriptome database of I. rubescens. Expressions of the genes in tissues were analyzed by RT-qPCR. Result (1) A total of 12 IrTIFYs genes were identified in I. rubescens. (2) The amino acid length was 124—378, the molecular weight 13 924.89—39 692.38 Da, and the isoelectric point ranged from 5.05 to 9.69. All members were unstable proteins, except for IrTIFY10. IrTIFY proteins were all located in the nucleus and were hydrophilic proteins without signal peptides. (3) Structural analysis indicates that IrTIFY proteins lack transmembrane structure and that the most abundant secondary structure type is random coil. Furthermore, all TIFY proteins contain multiple phosphorylation sites. (4) The IrTIFY gene family had weak codon preference, with a slight tendency to use codons ending in A or U. (5) There were many light-, hormone-, and stress-responsive cis-elements in the IrTIFY gene family, but cis-elements were difference in numbers and types among different members.（6）Evolutionary tree analysis showed that the 12 members of the TIFY family were divided into four subfamilies: PPD (IrTIFY2), ZML (IrTIFY3/8/10), TIFY (IrTIFY7/12), and JAZ (IrTIFY1/4/5/6/9/11). They were closest to that of Salvia miltiorrhiza of Labiaceae family. (7) RT-qPCR analysis revealed that the expression of all 12 members of the TIFY family of I. rubescens in different tissues was as follows: leaves > stems > roots, and most of them were significantly different. Conclusion Based on the above results, it is hypothesised that the TIFY gene family plays an important regulatory role in the growth and development of I. rubescens and may be involved in the regulation of the synthesis of secondary metabolites of I. rubescens, which lays the foundation for further in-depth study of the function of the TIFY gene family in I. rubescens and provides an idea for the further study of the function of the TIFY gene family in I. rubescens.
- Isodon rubescens /
- TIFY gene family /
- bioinformatics analysis /
- expression analysis

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图 1 冬凌草TIFY家族蛋白三级结构预测分析

Figure 1. Predicted tertiary structure of TIFY family proteins in I. rubescens

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图 2 冬凌草TIFY家族蛋白保守结构域比对分析

Figure 2. Alignment analysis on conserved domain of TIFY family proteins of I. rubescens

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图 3 冬凌草TIFY家族蛋白结构域可视化分析

Figure 3. Visualization of conserved protein domain of TIFY family proteins in I. rubescens

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图 4 冬凌草TIFY家族蛋白保守基序

Figure 4. Schematic conserved motif of TIFY family proteins in I. rubescens

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图 5 冬凌草TIFY家族成员启动子元件分析

Figure 5. Cis-elements analysis of TIFY family promoters in I. rubescens

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图 6 冬凌草、拟南芥、丹参和水稻TIFY家族成员的系统树分析

Ir：冬凌草；At：拟南芥；Sm：丹参；Os：水稻；Ptr：杨树。

Figure 6. Phylogenetic trees of TIFY family members in I. rubescens, A. thaliana, S. miltiorrhiza, O. sativa, and P. trichocarpa

Ir：I. rubescens；At：Arabidopsis thaliana；Sm：Salvia miltiorrhiza；Os：Oryza sativa; Ptr: Populus trichocarpa.

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图 7 冬凌草TIFY家族成员的不同组织表达分析

不同小写字母代表在 0.05 水平差异显著。

Figure 7. Expressions of TIFYs in different tissues of I. rubescens

Data with different lowercase letters represent significant differences(P＜0.05).

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表 1 荧光定量 PCR 引物序列

Table 1. Primer sequences for RT-qPCR

基因名称 Gene name	正向引物序列（5′-3′） Forward primer sequence （5′-3′）	反向引物序列（5′-3′） Reverse primer sequence （5′-3′）
UBQ	CTGAGGCTTCGTGGAGGGAT	TGTAGTCTGCAAGAGTCCTT
IrTIFY1	TGTCACCCCTTTCGTTCACC	AGTCATCGGAGGGCCATTTC
IrTIFY2	TTGATAAGCCGCTCCACCAG	TTTCCGCCTCACCAGTTGTT
IrTIFY3	CGAGAACTGGGATTCAGGCA	TGTTCGCCCACATTAACCCA
IrTIFY4	ACGGAAAGCATCTCTGGCTC	ACCTGCATTCTTGCTCTGGT
IrTIFY5	ACGCTTGGCTTCACTCCTAC	CCTCGGGCTTAATCTGGGTC
IrTIFY6	GCCGCTCCAAATTCAGAGGA	TCCGGAGGGTTCAACTACCA
IrTIFY7	GCGATCATATCGTTGGCGAG	GCGATCTCTTCATCGACAGC
IrTIFY8	GGGGAACAGATGGCAATGGA	CGGCGCATCATTGGAGTAGA
IrTIFY9	AGGAGGCAACCAGGGAAATC	CATTGAGGGCTGGGGTTGAG
IrTIFY10	TATGATGCGACGTGGACCAG	TAATGGCGTTTGCAGGAGGT
IrTIFY11	ATTACGTCGAGGGCCATTCC	ACTCGTGCTCGGGTTCATTT
IrTIFY12	TGGGTAGTTTGAGCAGGCAG	CCGGCTCCAACTCCAATCAA

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表 2 冬凌草TIFY家族蛋白理化性质分析

Table 2. Physicochemical properties of TIFY family proteins in I. rubescens

基因名称 Gene name	转录组基因ID Gene ID in transcriptome	长度 Length/aa	分子量 Molecular weight/Da	等电点 Isoelectric point	不稳定指数 Instability index	脂肪族指数 Aliphatic index	亲水性平均值 Grand average of hydropathicity
IrTIFY1	D-tissues_transcript_12000	356	37,515.67	9.01	58.96	76.97	−0.169
IrTIFY2	D-tissues_transcript_12412	344	37,381.18	8.09	50.35	69.22	−0.633
IrTIFY3	D-tissues_transcript_13824	343	37,606.01	5.05	49.03	66.56	−0.567
IrTIFY4	D-tissues_transcript_14652	299	31,555.55	8.59	56.46	58.83	−0.438
IrTIFY5	D-tissues_transcript_15941	229	24,182.28	7.81	53.98	72.71	−0.315
IrTIFY6	D-tissues_transcript_16425	173	18,692.28	8.99	41.53	75.78	−0.409
IrTIFY7	D-tissues_transcript_17017	124	13,924.89	9.69	66.65	77.26	−0.473
IrTIFY8	D-tissues_transcript_27842	299	32,514.88	6.17	40.31	58.66	−0.819
IrTIFY9	D-tissues_transcript_48324	250	27,235.7	8..93	68.06	66.4	−0.634
IrTIFY10	D-tissues_transcript_51903	300	32,486.03	5.63	37.71	59.2	−0.761
IrTIFY11	D-tissues_transcript_53470	273	29,436.81	9.15	56.22	65.79	−0.778
IrTIFY12	D-tissues_transcript_57396	378	39,692.38	9.22	46.47	46.8	−0.815

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表 3 冬凌草TIFY家族蛋白二级结构分析

Table 3. Secondary structure of TIFY family proteins in I. rubescens

基因名称 Gene name	转录组基因ID Gene ID in transcriptome	α-螺旋 α-helix/%	延伸链 Extension/%	β-转角 β-angle/%	无规则卷曲 Random curling/%
IrTIFY1	D-tissues_transcript_12000	11.24	12.36	2.53	73.88
IrTIFY2	D-tissues_transcript_12412	19.77	7.56	3.2	69.48
IrTIFY3	D-tissues_transcript_13824	22.45	10.79	5.83	60.93
IrTIFY4	D-tissues_transcript_14652	12.04	11.37	2.68	73.91
IrTIFY5	D-tissues_transcript_15941	17.9	12.66	4.37	65.07
IrTIFY6	D-tissues_transcript_16425	23.12	13.29	5.78	57.8
IrTIFY7	D-tissues_transcript_17017	27.56	11.81	5.51	55.12
IrTIFY8	D-tissues_transcript_27842	19.06	11.71	4.68	64.55
IrTIFY9	D-tissues_transcript_48324	20	7.6	4	68.4
IrTIFY10	D-tissues_transcript_51903	18	13.33	4.67	64
IrTIFY11	D-tissues_transcript_53470	13.92	6.59	2.93	76.56
IrTIFY12	D-tissues_transcript_57396	7.41	12.43	2.65	77.51

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表 4 冬凌草TIFY家族蛋白磷酸化位点、信号肽、跨膜结构和亚细胞定位分析

Table 4. Phosphorylation site, signal peptide, transmembrane structure, and subcellular localization of TIFY family proteins in I. rubescens

基因名称 Gene name	磷酸化位点 Phosphorylation sites	丝氨酸位点 Serine site/个	苏氨酸位点 Threonine site/个	酪氨酸位点 Tyrosine site/个	信号肽值 Signal peptides value	有无信号肽 Signal peptides	跨膜位置 Trans membrane localization	跨膜结构 Trans- membrane domain	亚细胞定位 Subcellular localization
IrTIFY1	54	32	20	2	0.001	无 No	膜外 Outside	0	细胞核 Nuclear
IrTIFY2	44	25	16	3	0.0007	无 No	膜外 Outside	0	细胞核 Nuclear
IrTIFY3	38	20	17	1	0.0007	无 No	膜外 Outside	0	细胞核 Nuclear
IrTIFY4	36	29	6	1	0.0005	无 No	膜外 Outside	0	细胞核 Nuclear
IrTIFY5	20	18	2	0	0.0013	无 No	膜外 Outside	0	细胞核 Nuclear
IrTIFY6	18	14	4	0	0.0018	无 No	膜外 Outside	0	细胞核 Nuclear
IrTIFY7	18	13	4	1	0.0014	无 No	膜外 Outside	0	细胞核 Nuclear
IrTIFY8	27	18	9	0	0.0011	无 No	膜外 Outside	0	细胞核 Nuclear
IrTIFY9	32	25	5	2	0.0012	无 No	膜外 Outside	0	细胞核 Nuclear
IrTIFY10	33	20	10	2	0.0022	无 No	膜外 Outside	0	细胞核 Nuclear
IrTIFY11	38	32	6	0	0.0035	无 No	膜外 Outside	0	细胞核 Nuclear
IrTIFY12	59	41	16	2	0.0011	无 No	膜外 Outside	0	细胞核 Nuclear

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表 5 冬凌草TIFY家族蛋白密码子偏好性分析

Table 5. Codon bias of TIFY family proteins in I. rubescens

基因名称 Gene name	转录组基因号 Gene ID in transcriptome	有效密码子数 ENC/个	GC/%	GC1s/%	GC2s/%	GC3s/%
IrTIFY1	D-tissues_transcript_12000	50.869	44.06	47.06	44.85	40.26
IrTIFY2	D-tissues_transcript_12412	55.717	46.92	45.85	50.19	44.72
IrTIFY3	D-tissues_transcript_13824	59.099	45.66	46.24	42.37	48.39
IrTIFY4	D-tissues_transcript_14652	54.066	43.83	43.44	39.62	48.45
IrTIFY5	D-tissues_transcript_15941	55.686	48.84	47.13	52.27	47.13
IrTIFY6	D-tissues_transcript_16425	52.629	46.55	44.21	44.21	51.23
IrTIFY7	D-tissues_transcript_17017	54.526	43.25	39.22	37.93	52.59
IrTIFY8	D-tissues_transcript_27842	58.699	44.06	42.25	48.49	41.45
IrTIFY9	D-tissues_transcript_48324	50.617	40.31	37.7	42.67	40.58
IrTIFY10	D-tissues_transcript_51903	53.436	49.66	50.77	50.26	47.96
IrTIFY11	D-tissues_transcript_53470	56.905	43.92	42.18	41.19	48.39
IrTIFY12	D-tissues_transcript_57396	60.714	46.35	49.59	38.39	51.07

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