小麦衰老相关基因<i>TaSAG39</i>的生物信息学及表达模式分析

doi:10.7668/hbnxb.20192703

摘要/Abstract

摘要：

半胱氨酸蛋白酶是蛋白质降解过程中的一类水解酶,参与植物的衰老与成熟,在植物生长发育过程中发挥重要作用。基于前期得到的小麦衰老阶段转录组数据中筛选到一个衰老特异的半胱氨酸蛋白酶(SAG39),为了研究TaSAG39基因在小麦衰老过程中发挥的作用,采用生物信息学分析方法和实时荧光定量(qRT-PCR)技术对TaSAG39的基因结构和表达模式进行分析。结果表明,其氨基酸序列与小麦祖先种节节麦、野生二粒小麦以及硬粒小麦亲缘关系最近,含有木瓜蛋白酶亚家族特有的活性位点Cys-His-Asn以及EFNIN结构。TaSAG39-5A、TaSAG39-5B、TaSAG39-5D基因组全长分别为1 455,1 435,1 439 bp,编码序列长分别为1 041,1 050,1 038 bp,均含2个外显子和1个内含子;其编码的蛋白分别由346,349,345个氨基酸组成,分子质量约37 ku,等电点5.53~5.67,为稳定的带负电的亲水性蛋白;主要构成元件为无规则卷曲和α-螺旋,在蛋白质N段含有信号肽,具有保守的Inhibitor_I29和Peptidase_C1结构域,含有35~37个磷酸化位点,其中以丝氨酸磷酸化位点和苏氨酸磷酸化位点为主。TaSAG39蛋白可能与半胱氨酸蛋白酶抑制剂以及14-3-3蛋白互作,该基因启动子中含有脱落酸、茉莉酸甲酯、生长素、光、干旱、低温以及胁迫响应等有关的顺式作用元件。qRT-PCR结果证实,TaSAG39基因受自然衰老、黑暗、干旱、茉莉酸甲酯、高温、生长素诱导表达,在自然衰老过程中基因的诱导表达最为显著。

关键词: 小麦, TaSAG39, 半胱氨酸蛋白酶, 生物信息学分析, 表达模式

Abstract:

Cysteine protease,a kind of hydrolase in the process of protein degradation,participates in the senescence and maturation of plants and plays an important role in the growth and development of plants.In this study,a senesence-specific cysteine protease(SAG39)was screened from the previously obtained transcriptome data of wheat at senescence stage.In order to study the role of TaSAG39 gene in wheat at senescence,the gene structure and expression pattern of TaSAG39 were analyzed by bioinformatics analysis and Real-time fluorescence quantitative(qRT-PCR)technology.The results showed that the amino acid sequence encoded by the cysteine protease gene SAG39 had the closest relationship with the ancestral species of wheat,such as Aegilops tauschii,Triticum dicoccoides and Triticum durum,and it included the unique active site Cys-His-Asn and EFNIN structures of the papain sub-family.The full-length genomes of TaSAG39-5A,TaSAG39-5B and TaSAG39-5D were 1 455,1 435,1 439 bp,respectively,and the length of the coding sequence was 1 041,1 050,1 038 bp,respectively,with each containing two exons and one intron.The TaSAG39s proteins consisted of 346,349,345 amino acids.With relative molecular weight of about 37 ku and isoelectric point of 5.53—5.67,these proteins were stable negatively charged hydrophilic proteins.Its main components were random coils and α-helix,and there were signal peptides in the N segment of the protein.It had the conserved Inhibittor_I29 and Petidase_C1 domains,and contained 35—37 phosphorylation sites,mainly serine and threonine phosphorylation sites.TaSAG39 protein may interact with cysteine protease inhibitors and 14-3-3 protein.The promoter contained cis-acting elements relative to abscisic acid,methyl jasmonate,auxin,light,drought,low temperature and stress response.The results of qRT-PCR confirmed that gene TaSAG39 was induced to express by natural aging,darkness,drought,methyl jasmonate,high temperature and auxin,among which natural aging was the most significant.

Key words: Wheat, TaSAG39, Cysteine protease, Bioinformatics analysis, Expression pattern

孙丽丽, 晋秀娟, 赵锴, Md Ashraful Islam, 卢娟, 王曙光, 孙黛珍. 小麦衰老相关基因TaSAG39的生物信息学及表达模式分析[J]. 华北农学报, 2022, 37(2): 18-27. doi: 10.7668/hbnxb.20192703.

SUN Lili, JIN Xiujuan, ZHAO Kai, Md Ashraful Islam, LU Juan, WANG Shuguang, SUN Daizhen. Bioinformatics and Expression Pattern Analysis of Senescence-associated Gene TaSAG39 in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 18-27. doi: 10.7668/hbnxb.20192703.

http://www.hbnxb.net/CN/Y2022/V37/I2/18

图/表 14

图1 TaSAG39蛋白系统进化树分析

Fig.1 Phylogenetic tree analysis of TaSAG39 protein

图2 TaSAG39氨基酸序列比对分析

^*.二硫键位点;黄色方框.EFNTIN结构;蓝色方框.半胱氨酸蛋白酶活性(Cys-His-Asn);红色线条.保守结构域Inhibitor_I29和Peptidase_C1。

Fig.2 Comparative analysis of TaSAG39 amino acid sequences

^*.The disulfide bond site;The yellow box.The structure of EFNIN;The blue box.The cysteine protease activity(Cys-His-Asn); The red line.The conserved domain Inhibitor_I29 and Peptidase_C1.

图3 TaSAG39氨基酸组成

Fig.3 Amino acid composition of TaSAG39

表1 TaSAG39蛋白质理化性质

Tab.1 Physicochemical properties of TaSAG39 protein

项目Item	TaSAG39-5A	TaSAG39-5B	TaSAG39-5D
氨基酸数量/个 Number of amino acids	346	349	345
分子质量/ku Molecular weight	36.67	36.90	36.53
不稳定系数Instability index	35.99	38.07	34.59
脂肪指数Aliphatic index	72.63	71.43	71.13
平均亲水性Grand average of hydropathicity	-0.183	-0.122	-0.174
理论等电点Theoretical Pi	5.53	5.67	5.62
带正电残基(Arg + Lys) Positively charged residues	36	35	35
带负电残基(Asp + Glu) Negatively charged residues	42	41	40
分子式Formula	C₁₅₇₈H₂₄₇₃N₄₇₃O₅₀₃S₁₇	C₁₅₀₅H₂₄₈₈N₄₇₄O₄₉₉S₁₉	C₁₅₇₆H₂₄₅₇N₄₇₁O₄₉₉S₁₇

图4 TaSAG39蛋白质二级结构预测

Fig.4 Prediction of secondary structure of TaSAG39 protein

图5 TaSAG39蛋白质三级结构预测

Fig.5 Prediction of tertiary structure of TaSAG39 protein

图6 TaSAG39蛋白信号肽预测

Fig.6 Prediction of signal peptide of TaSAG39 protein

图7 TaSAG39蛋白磷酸化位点预测

Fig.7 Prediction of phosphorylation site of TaSAG39 protein

图8 TaSAG39蛋白的保守基序分析

Fig.8 Conserved motif analysis of TaSAG39 protein

图9 TaSAG39蛋白的保守结构域

Fig.9 Conserved domains of TaSAG39 protein

图10 TaSAG39的基因结构分析

Fig.10 Analysis of TaSAG39 gene's structure

图11 TaSAG39蛋白互作分析

Fig.11 The analysis of TaSAG39 protein interaction networks

图12 TaSAG39启动子区顺式作用元件

Fig.12 Cis-acting elements in the promoter region of TaSAG39

图13 TaSAG39在不同处理下的表达模式
不同小写字母表示不同样本之间TaSAG39表达量的显著性差异(P<0.05)。

Fig.13 Expression pattern of TaSAG39 under different treatments
Different lowercase letters indicate significant difference in the expression of TaSAG39 gene in different samples at 0.05 level.

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小麦衰老相关基因TaSAG39的生物信息学及表达模式分析

Bioinformatics and Expression Pattern Analysis of Senescence-associated Gene TaSAG39 in Wheat

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