广藿香Trihelix家族<i>PatASIL2</i>基因克隆、亚细胞定位及表达分析

doi:10.7668/hbnxb.20195335

摘要/Abstract

摘要：

为探究广藿香Trihelix家族基因PatASIL2的序列特征、亚细胞定位及表达模式,以广藿香转录组获得的ASIL2基因序列设计特异性引物,以广藿香cDNA为模板,克隆PatASIL2基因,随后对其进行生物信息学分析;构建PatASIL2-EGFP融合蛋白表达载体并转化拟南芥原生质体,检测PatASIL2的亚细胞定位;利用实时荧光定量PCR(qRT-PCR)分析PatASIL2基因在广藿香不同组织、外源茉莉酸甲酯(MeJA)喷施及非生物胁迫(盐胁迫、干旱胁迫、冷胁迫)下的表达情况。结果显示,PatASIL2含长度为1 035 bp的开放阅读框,编码344个氨基酸。PatASIL2不具有跨膜结构和信号肽,属于不稳定的亲水蛋白,含有41个丝氨酸磷酸化位点和1个Myb_DNA-bind_4保守结构域。系统进化分析显示,PatASIL2归类到Trihelix转录因子家族的SIP1亚家族,与芝麻SiASIL2同源性较高。亚细胞定位结果表明,PatASIL2为细胞核定位蛋白。qRT-PCR结果表明,PatASIL2在广藿香的嫩叶、成熟叶、老叶、根和茎中都有表达,尤其在老叶中的表达量最高;在MeJA处理后,PatASIL2基因的表达量在12~24 h显著升高;盐胁迫下,PatASIL2基因的表达量在3~24 h显著升高;在干旱胁迫下,PatASIL2基因的表达量在24 h显著升高;在冷胁迫下,PatASIL2基因在12 h表达量显著升高。

关键词: 广藿香, Trihelix, ASIL2, 基因克隆, 表达模式, 亚细胞定位

Abstract:

To reveal the sequence characteristics,subcellular localization,and expression pattern of PatASIL2,belonging to the Trihelix transcription factor family,the PatASIL2 gene was first cloned using the cDNA of Pogostemon cablin (patchouli)as a template,and the bioinformatics analysis was further performed.The PatASIL2-EGFP expressed vector was constructed and transformed into Arabidopsis protoplasm to investigate the subcellular localization of PatASIL2.The real-time quantitative reverse transcription PCR(qRT-PCR)was also performed to detect the expression profiles of PatASIL2 gene in different tissues of patchouli and under treatments of methyl jasmonate(MeJA),salt stress,drought stress,and cold stress.The results indicated that PatASIL2 gene contained an open reading frame of 1 035 bp and encoded 344 amino acids.The PatASIL2 protein was an unstable hydrophilic protein without transmembrane domains and signal peptides and had 41 serine phosphorylation sites and one Myb_DNA-bind_4 conserved domain.The phylogenetic analysis showed that PatASIL2 was classified into the SIP1 subfamily of Trihelix transcription factor family,and clustered closely with Sesamum indicum SiASIL2.The subcellular localization result indicated that PatASIL2 was a nuclear-localized protein.The qRT-PCR results indicated that PatASIL2 expressed in the young leaf,mature leaf,old leaf,stem,and root in patchouli,particularly with the highest expression in the old leaf.The PatASIL2 expression was significantly upregulated by MeJA at 12—24 h and salt stress at 3—24 h.The expression of PatASIL2 was significantly upregulated at 24 h after drought treatment,and at 12 h after cold treatment.

Key words: Pogostemon cablin, Trihelix, ASIL2, Gene cloning, Expression pattern, Subcellular localization

李俊仁, 陈秀珍, 吴带娣. 广藿香Trihelix家族PatASIL2基因克隆、亚细胞定位及表达分析[J]. 华北农学报, 2025, 40(1): 104-112. doi: 10.7668/hbnxb.20195335.

LI Junren, CHEN Xiuzhen, WU Daidi. Cloning,Subcellular Localization,and Expression Analysis of PatASIL2 Gene of Trihelix Family from Pogostemon cablin[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(1): 104-112. doi: 10.7668/hbnxb.20195335.

http://www.hbnxb.net/CN/Y2025/V40/I1/104

图/表 10

表1 生物信息学分析软件及用途

Tab.1 Bioinformatics analysis software and its applications

分析内容 Analysis content	软件名称 Software name	网址 Website
保守结构域 Conservative structural domain	NCBI-CDD	https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
理化性质 Physical and chemical properties	ProtParam	https://web.expasy.org/protparam/
亲/疏水性 Pro/hydrophobic	ProtScale	https://web.expasy.org/protscale/
亚细胞定位 Subcellular localization	WoLF PSORT	https://wolfpsort.hgc.jp/
信号肽 Signal peptide	SignalP-4.1	https://services.healthtech.dtu.dk/service.php?SignalP-4.1
跨膜区 Transmembrane domain	TMHMM 2.0	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0
磷酸化位点 Phosphorylation site	NetPhos-3.1	https://services.healthtech.dtu.dk/services/NetPhos-3.1/
二级结构 Secondary structure	PSIPRED	http://bioinf.cs.ucl.ac.uk/psipred/
三级结构 Tertiary structure	SWISS-MODEL	https://swissmodel.expasy.org/

图1 广藿香PatASIL2基因的PCR扩增电泳条带(A)和cDNA序列(B)

Fig.1 Agarose gel electrophoresis of PCR amplification(A)and cDNA sequence(B)of patchouli PatASIL2

图2 广藿香PatASIL2和拟南芥Trihelix家族蛋白的系统进化分析

Fig.2 The phylogenetic analysis of PatASIL2 and Arabidopsis thaliana Trihelix family proteins

图3 PatASIL2的蛋白亲水性/疏水性(A)、蛋白信号肽(B)、蛋白保守结构域(C)、蛋白跨膜结构域(D)及蛋白磷酸化位点(E)预测

Fig.3 Prediction of hydrophilicity/hydrophobicity(A),protein signal peptide(B),conservative domain of protein(C), protein transmembrane domain(D),and protein phosphorylation site(E)of PatASIL2

图4 PatASIL2蛋白二级结构(A)和三级结构(B)预测

Fig.4 Prediction of secondary structure(A)and tertiary structure(B)of PatASIL2

图5 广藿香和其他植物ASIL2氨基酸的多序列比对

Fig.5 Amino acid sequence alignment of Trihelix transcription factor ASIL2 in patchouli and other plant species

图6 广藿香与其他植物ASIL2蛋白的系统进化分析

Fig.6 The phylogenetic analysis of Trihelix transcription factor ASIL2 in patchouli and other plant species

图7 pAN580-PatASIL2表达载体简图(A)及PatASIL2的亚细胞定位(B)

Fig.7 Schematic representation of the pAN580-PatASIL2 construct(A)and the subcellular localization of PatASIL2(B)

图8 广藿香PatASIL2基因在不同组织中的相对表达量不同小写字母代表不同组织间差异显著(P<0.05)。

Fig.8 Relative expression levels of PatASIL2 in different tissues of patchouli The different lowercase letters represent significant differences in different tissues(P<0.05).

图9 广藿香PatASIL2基因在茉莉酸甲酯处理(A)、盐胁迫(B)、干旱胁迫(C)及冷胁迫(D)下的表达模式不同小写字母代表不同时间点间差异显著(P<0.05)。

Fig.9 Expression profiles of PatASIL2 under treatments of MeJA(A),salt(B),drought(C),and cold(D) The different lowercase letters represent significant differences in different time points(P<0.05).

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广藿香Trihelix家族PatASIL2基因克隆、亚细胞定位及表达分析

Cloning,Subcellular Localization,and Expression Analysis of PatASIL2 Gene of Trihelix Family from Pogostemon cablin

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广藿香Trihelix家族PatASIL2基因克隆、亚细胞定位及表达分析

Cloning,Subcellular Localization,and Expression Analysis of PatASIL2 Gene of Trihelix Family from Pogostemon cablin

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