基于冬凌草全长转录组的TIFY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

基于冬凌草全长转录组的TIFY基因家族鉴定与表达分析

1.
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，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（豫农科教[2018]14号）；2019年中医药公共卫生服务补助专项“全国中药资源普查项目”（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

Identification and Expression Analysis of TIFY Gene Family in Isodon rubescens

1.
School of Pharmacy, Henan Key Laboratory of Chinese Medicine Resources and Chemistry/Henan University of Chinese Medicine, Zhengzhou, Henan　450046, China

摘要

摘要: 目的 TIFY 蛋白是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 技术分析其不同组织中的表达特性。结果（1）成功从冬凌草中共鉴定出12个TIFY家族基因成员；（2）理化性质分析表明其氨基酸长度范围是124～378 aa；分子量大小为13 924.89～39 692.38 Da；等电点的范围5.05～9.69；除IrTIFY10为稳定蛋白，其他均为不稳定蛋白；IrTIFYs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。结论基于以上结果，推测TIFY基因家族在对冬凌草的生长发育过程中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思路。
- 冬凌草 /
- TIFY基因家族 /
- 生物信息学分析 /
- 表达分析
Abstract: Objective TIFY proteins are key regulators of JA signaling pathway, which also play significant regulatory roles in plant growth and development, abiotic stress, and secondary metabolites accumulation. Study the TIFY genes of Isodon rubescens can provide a theoretical basis for the improvement breeding of stress resistance and the synthesis of secondary metabolites in I. rubescens. Method In this study, the TIFY gene family was identified and analyzed by bioinformatics methods based on the full-length transcriptome database of I. rubescens, and the expression characteristics of the TIFY gene family in different tissues were analyzed by RT-qPCR. Result (1) A total of 12 TIFY genes were identified in the full-length transcriptome data of I. rubescens. (2) Physicochemical properties analysis showed that the amino acid length of IrTIFYs ranged from 124 aa to 378 aa. The molecular weight ranges from 13924.89 to 39692.38 Da. The isoelectric points were rang from 5.05 to 9.69; All the proteins were belong to stable proteins except IrTIFY10. The subcellular location analysis showed that all IrTIFY proteins were located in the nucleus, and all TIFYs are belong to hydrophilic proteins without signal peptides. (3) The structural analysis showed that none of the IrTIFY proteins had a transmembrane structure, and random curling was the most abundant structure type in the secondary structure. All proteins contains multiple phosphorylation sites. (4) Codon preference analysis showed that the codon preference of IrTIFY gene family was weak, and the codon ending with A or U was slightly inclined to be used. (5) The cis-elements analysis of IrTIFYs promoter showed that there were many light responsive cis-elements, hormone responsive cis-elements, and stress responsive cis-elements in the TIFY family. However, there were differences in the numbers and types of cis-elements among different IrTIFY members.（6）The phylogenetic tree analysis showed that 12 IrTIFYs were divided into four subfamilies: PPD (IrTIFY2), ZML (IrTIFY3/8/10), TIFY (IrTIFY7/12) and JAZ (IrTIFY1/4/5/6/9/11). and IrTIFYs had the closest genetic relationship with Salvia miltiorrhiza, which also belong to the labiaceae family. (7) RT-qPCR analysis showed that the expression levels of 12 TIFYs exhibited same trend with leaf ＞ stem ＞ root of different tissues, and most of the members showed significant differences between different tissues. Conclusion Based on the above results, it is speculated that IrTIFYs play an important regulatory role in the growth and development of I. rubescens, and may be involved in regulating the biosynthesis of secondary metabolites of I. rubescens, which lays a foundation and provides ideas for further research on the function of 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

(A) IrTIFY1蛋白；(B) IrTIFY2蛋白；(C) IrTIFY3蛋白；(D) IrTIFY4蛋白；(E) IrTIFY5蛋白；(F) IrTIFY6蛋白；(G) IrTIFY7蛋白；(H) IrTIFY8蛋白；(I) IrTIFY9蛋白；(J) IrTIFY10蛋白；(K) IrTIFY11蛋白；(L) IrTIFY12蛋白

Figure 1. Tertiary structure prediction analysis of TIFY protein family in I. rubescens

(A) IrTIFY1 protein；(B) IrTIFY2 protein；(C) IrTIFY3 protein；(D) IrTIFY4 protein；(E) IrTIFY5 protein；(F) IrTIFY6 protein；(G) IrTIFY7 protein；(H) IrTIFY8 protein；(I) IrTIFY9 protein；(J) IrTIFY10 protein；(K) IrTIFY11 protein；(L) IrTIFY12 protein

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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 of the 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 analysis of TIFY protein family in I. rubescens

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图 4 冬凌草TIFY家族蛋白保守基序

Figure 4. Schematic diagram of conserved protein motif analysis of TIFY protein family 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-element analysis of TIFYs promoters in I. rubescens

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图 6 冬凌草TIFY家族系统树分析

Ir：冬凌草；At：拟南芥；Sm：丹参；Os：水稻。

Figure 6. Phylogenetic trees of TIFY gene family in I. rubescens

Ir：Isodon 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

小写字母代表在 0.05 水平上差异显著。

Figure 7. Expressions of TIFYs in different organs of I. rubescens plant

Data with lowercase letters represent significant differences at P＜0.05.

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表 1 荧光定量 PCR 引物序列

Table 1. Primer sequences for fluorescent quantitative PCR

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 protein family 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 analysis of TIFY protein family 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 analysis of TIFY protein family in I. rubescens

基因名称 Gene name	磷酸化位点 Phosphorylation sites	丝氨酸 Serine	苏氨酸 Threonine	酪氨酸 Tyrosine	信号肽值 Signal peptides value	有无信号肽 Signal peptides	跨膜位置 Trsns membrane localization	跨膜结构数 Trsns 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 analysis of TIFY protein family 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	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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