Bioinformatics and Expression of Glycerol-3-phosphate Acyltransferase 13 Gene of Maize
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摘要:
目的 对玉米（Zea mays L.）甘油-3-磷酸酰基转移酶（Glycerol-3-phosphate acyltransferase，GPAT）家族成员ZmGPAT13进行生物信息学分析和在不同非生物胁迫处理下的基因表达模式变化检测，为进一步研究ZmGPAT13的生物学功能奠定理论基础。 方法 通过TAIR和MaizeGDB数据库获得拟南芥（Arabidopsis thaliana）和玉米中GPAT家族蛋白序列，利用生物信息学方法进行理化性质、蛋白结构、亚细胞定位、保守基序、系统进化、磷酸化位点和启动子顺式作用元件等分析，并采用荧光定量PCR（qRT-PCR）技术对ZmGPAT13在不同组织部位及甘露醇（Mannitol）、NaCl、脱落酸（ABA）、高温（42 ℃）、低温（4 ℃）处理下的相对基因表达量进行分析。 结果 ZmGPAT13是一个定位于线粒体的跨膜蛋白，与部分GPAT家族成员含有排序相同的15个保守基序（motif），且与拟南芥中AtGPAT1的亲缘关系最近；预测分析发现ZmGPAT13含有62个磷酸化位点，可能与玉米中多个GPATs存在相互作用关系；ZmGPAT13基因启动子富含多种与低温和ABA响应等相关的顺式作用元件。通过qRT-PCR技术对ZmGPAT13组织表达模式分析发现ZmGPAT13的相对基因表达量在胚芽鞘（Coleoptile）中最高，根（Root）中次之，叶片（Leaf）中最低；甘露醇（Mannitol）、NaCl、脱落酸（ABA）及高温（42 ℃）和低温（4 ℃）处理均能诱导ZmGPAT13的基因表达显著上调。 结论 玉米中ZmGPAT13具有高度的进化保守性，可能通过转录水平的变化参与调控玉米对干旱、盐、高温和低温等非生物胁迫的响应过程。 -
关键词:
- 玉米 /
- 甘油-3-磷酸酰基转移酶 /
- ZmGPAT13 /
- 生物信息学分析 /
- 非生物胁迫
Abstract:Objective Bioinformatics and expressions under stresses of glycerol-3-phosphate acyltransferase (GPAT) gene family member, ZmGPAT13, in maize were studied. Method Sequences of GPAT family members in Arabidopsis thaliana and Zea mays L. were obtained from the TAIR and MaizeGDB databases. Physicochemical properties, protein structure, subcellular localization, conserved motifs, phylogenetic evolution, phosphorylation sites, and cis-acting elements of promoter sequence of the GPATs were analyzed by bioinformatic methods. Expressions of ZmGPAT13under the stresses of mannitol, NaCl, abscisic acid (ABA), high temperature at 42 ℃, and low temperature at 4 ℃ were examined by qRT-PCR. Result A transmembrane protein, ZmGPAT13 localized in mitochondria, shared the same 15 conserved motifs with other GPAT family members, and had the highest homology with AtGPAT1 in arabidopsis. It contained 62 phosphorylation sites and might interact with multiple GPATs of maize. The promoters of ZmGPAT13consisted of numerous cis-acting elements associated with the responses to low temperature and ABA stresses. The gene expressed most highly in the coleoptile, followed by the roots and the lowest in the leaves. And, relatively, the expressions were significantly elevated under the stress of mannitol, NaCl, ABA or high or low temperature. Conclusion A high evolutionary conservation seemed existed in ZmGPAT13. It was postulated that, at transcription level, ZmGPAT13 might be involved in the responses of maize plants to abiotic stresses such as drought, salt, and extreme temperatures. -
Key words:
- Zea mays L. /
- glycerol-3-phosphate acyltransferase /
- ZmGPAT13 /
- bioinformatics /
- abiotic stresses
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图 1 ZmGPAT13跨膜结构域（A）、蛋白二级结构（B）和蛋白三级结构（C）分析
Figure 1. Transmembrane domain (A), secondary structure (B), and tertiary structure (C) of ZmGPAT13
图 2 拟南芥和玉米中GPAT家族成员保守基序分析（A）及蛋白保守基序的氨基酸序列（B）
Figure 2. Conserved motifs of GPATs from arabidopsis and maize (A), and amino acid sequences of conserved motifs (B)
图 3 拟南芥和玉米中GPAT家族成员进化树分析
Figure 3. Phylogenetic tree of GPATs from arabidopsis and maize
图 4 玉米中ZmGPAT13在不同组织及不同外源非生物胁迫处理下的表达
A：不同玉米组织；B：200 mmol·L−1 甘露醇处理；C：200 mmol·L−1 NaCl处理；D：100 μmol·L−1 ABA处理；E：42 ℃高温处理；F：4 ℃低温处理。不同小写字母表示不同处理间差异显著（P ＜ 0.05）。
Figure 4. Relative expressions of ZmGPAT13 in tissues under abiotic stresses
A: Different tissues; B: Mannitol treatment at 200 mmol·L−1; C: NaCl treatment at 200 mmol·L−1; D: ABA treatment at 100 μmol·L−1; E: High-temperature treatment at 42 ℃; F: Low temperature treatment at 4 ℃. Data with different lowercase letters indicate significant differences at P＜0.05.
表 1 实时荧光定量PCR引物序列
Table 1. Primer pairs for qRT-PCR verification
基因
Gene引物
Primer引物序列（5′-3′）
Primer sequence(5′-3′)ZmGPAT13 ZmGPAT13-qF CGATCAGGGAGGCGTTGTTA ZmGPAT13 ZmGPAT13-qR CCTTGCAGTAGGGGACGAAG ZmActin2 ZmActin2-qF GCCATCCATGATCGGTATGG ZmActin2 ZmActin2-qR GTCGCACTTCATGATGGAGTTG 
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表 2 ZmGPAT13磷酸化位点预测分析
Table 2. Predicted phosphorylation sites of ZmGPAT13
氨基酸
Amino acid磷酸化位点
Phosphorylation sites激酶类型
Kinase groups丝氨酸 Serine 43(2)，187(2)，222(2)，279(2)，286(2)，353(2)，451(2)，496(2)，540(2) cdc2，PKC，PKG，RSK 51，104，168，221，223，257，258，276，324，397，402，479 DNAPK，cdc2，PKA，UNSP，CKII，PKC 苏氨酸 Threonine 37(2)，217(2)，227(2)，414(2)，472(2)，510(2)，516(2) PKC，UNSP，PKB，CKII，cdc2，PKG 17，38，55，60，65，89，180，385，440，441， 384，410，423 PKC，cdc2，UNSP 酪氨酸 Tyrosine 273，290，370，474，545 INSR，UNSP cdc2：细胞周期依赖性蛋白激酶；CKII：酪蛋白激酶II；DNAPK：DNA依赖性激酶；INSR：受体酪氨酸蛋白激酶；PKA：蛋白激酶A； PKC：蛋白激酶C；PKG：蛋白激酶G；RSK：核糖体S6蛋白激酶；PKB：蛋白激酶B；UNSP：无特异性蛋白激酶。括号内数字为所含氨基酸磷酸化位点个数。
cdc2: Cyclin-dependent kinase; CKII: Casein protein kinase II; DNAPK: DNA-dependent protein kinase; INSR: Insulin receptor tyrosine protein kinase; PKA: Protein kinase A; PKC: Protein kinase C; PKG: Protein kinase G; RSK: Ribosomal protein S6 kinases; PKB: Protein kinase B; UNSP: Unknown specific protein kinase. Number in brackets indicates count of amino acid sites contained.
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表 3 ZmGPAT13功能互作蛋白预测分析
Table 3. Predicted functional interaction proteins of ZmGPAT13
基因编号
Gene ID功能结构域
Function domain氨基酸
Amino
acid互作系数
Interaction
coefficientGRMZM2G123987 PlsC 371 0.954 GRMZM2G159890 PlsC，GPAT_N domain 477 0.940 GRMZM2G165681 PlsC 371 0.936 GRMZM2G135027 PlsC，Acyltransf_C domain 399 0.783 GRMZM2G014981 PlsC，Acyltransf_C domain 403 0.783 GRMZM2G037104 PlsC，Acyltransf_C domain 374 0.763 GRMZM2G878139 PlsC，Acyltransf_C domain 374 0.763 GRMZM2G071076 Choline_transpo domain 537 0.721 GRMZM2G075295 PlsC，HAD domain 486 0.698 GRMZM2G083195 PlsC，HAD domain 502 0.682
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表 4 ZmGPAT13基因启动子顺式作用元件分析
Table 4. Cis-acting elements of ZmGPAT13 promoter
序号
No.元件名称
Element序列
Sequence功能预测
Predicted function1 ABRE ACGTG 脱落酸响应
Abscisic acid responsive2 LTR CCGAAA 低温响应
Low temperature responsive3 TGACG-motif TGACG 茉莉酸甲酯响应
MeJA responsive4 CGTCA-motif CGTCA 茉莉酸甲酯响应
MeJA responsive5 Sp1 GGGCGG 光响应
Light responsive6 O2-site GATGATGTGG 玉米醇溶蛋白代谢
Zein metabolism7 GCN4_motif TGAGTCA 胚乳表达
Endosperm expression8 GC-motif CCCCCG 厌氧诱导
Anaerobic induction9 AuxRR-core GGTCCAT 生长素响应
Auxin responsive10 CCAAT-box CAACGG MYBHv1结合位点
MYBHv1 binding site
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