Identification and Expressions of TIFY Family in Isodon rubescens
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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。 Abstract:Objective Genes of Isodon rubescens relating to TIFY proteins were identified and expressions analyzed. 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) TwelveIrTIFYs were identified in I.rubescens. (2) The genes had 124-378 amino acids with a molecular weight ranging from 13,924.89-39,692.38Da and isoelectric points of 5.05-9.69. Located in the nucleus, the proteins were hydrophilic without signal peptides, and except IrTIFY10, unstable. (3) None of the proteins showed a transmembrane structure. Random coil was the most abundant secondary structure. All of them contained multiple phosphorylation sites. (4) The codon preference of IrTIFYs was weak and ended with A or U. (5) There were many light-, hormone-, and stress-responsive cis-elements in the family that differed in numbers and types among the members.(6)Phylogenetically, the 12 IrTIFYs belonged to 4 subfamilies of 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) The expressions of the genes were all highest in the leaves, in between in the stems, and lowest in the roots with significant differences. Conclusion It appeared that IrTIFYs played an important role in regulating the growth and development of I. rubescens. They might also be associated with the biosynthesis of secondary metabolites in the plant. -
Key words:
- Isodon rubescens /
- TIFY 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. Predicted tertiary structure of TIFYs 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.
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 表 2 冬凌草TIFY家族蛋白理化性质分析
Table 2. Physicochemical properties of TIFYs 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 hydropathicityIrTIFY1 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 表 3 冬凌草TIFY家族蛋白二级结构分析
Table 3. Secondary structure of TIFYs in I. rubescens
基因名称
Gene name转录组基因ID
Gene ID in transcriptomeα-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 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 of TIFYs 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
localizationIrTIFY1 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 表 5 冬凌草TIFY家族蛋白密码子偏好性分析
Table 5. Codon bias of TIFYs 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
ENCGC/% 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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