广藿香Trihelix转录因子家族基因<i>PatGTL1</i>的克隆、亚细胞定位及表达分析

doi:10.7668/hbnxb.20195190

摘要/Abstract

摘要：

为了明确广藿香Trihelix转录因子家族成员PatGTL1的序列特征及表达特性,并为后续深入研究PatGTL1转录因子的功能奠定理论基础。首先,以广藿香为材料提取总RNA并反转录得到cDNA,以此为模板根据转录组序列设计特异性引物克隆PatGTL1基因,随后利用生物信息学软件分析预测其理化性质、结构特征和亲缘关系;构建pAN580-PatGTL1-EGFP融合表达载体并转化拟南芥原生质体,考察PatGTL1亚细胞定位;进一步利用实时荧光定量PCR(qRT-PCR)测定PatGTL1基因在广藿香不同组织中、外源茉莉酸甲酯(MeJA)处理及非生物胁迫(包括盐胁迫、干旱胁迫、冷胁迫)下的表达模式。结果表明,PatGTL1开放阅读框为1 497 bp,编码498个氨基酸。PatGTL1为不稳定亲水蛋白,无跨膜结构和信号肽,含有42个丝氨酸磷酸化位点,含有2个GT1保守结构域。进化树分析表明,PatGTL1属于Trihelix转录因子家族的GT-2亚家族,与芝麻SiGTL1同源性较高。亚细胞定位结果表明,PatGTL1定位于细胞核。qRT-PCR分析显示,PatGTL1在广藿香的根、茎、嫩叶、成熟叶和老叶中均有表达,尤以嫩叶中的表达量最高;在MeJA处理后,PatGTL1的相对表达量显著提高,在3~24 h呈上升趋势;在盐胁迫3 h PatGTL1表达量最高;干旱胁迫后PatGTL1表达量显著低于对照;冷胁迫后PatGTL1表达量呈先上升后下降的趋势,在12 h表达量最高。研究结果丰富了广藿香Trihelix转录因子家族的研究。

关键词: 广藿香, Trihelix, GTL1, 基因克隆, 表达模式, 亚细胞定位

Abstract:

In order to elucidate the sequence characteristics and expression pattern of PatGTL1,a member of Trihelix transcription factor family,and lay a foundation for the further function study of PatGTL1,the total RNA of Pogostemon cablin (patchouli)was extracted and reverse-transcribed to cDNA.The specific primers were designed according to the transcriptome sequence of PatGTL1,and the cDNA was used as a template to clone PatGTL1 gene.The bioinformatics analysis was further performed to reveal the structural,physicochemical properties,and phylogenetic relationships of PatGTL1.Besides,the pAN580-PatGTL1-EGFP vector was built and transformed into Arabidopsis protoplastsa to detect the subcellular localization of PatGTL1.The Real-time Quantitative reverse transcription PCR(qRT-PCR)was also carried out to investigate the expression patterns of PatGTL1 gene in different tissues of patchouli and under treatments of methyl jasmonate(MeJA),salt stress,drought stress,and cold stress.The results showed that the open reading frame of the PatGTL1 gene was 1 497 bp,encoding 498 amino acids.The PatGTL1 protein was an unstable hydrophilic protein that contained 42 serine phosphorylation sites and was predicted to be located in the nucleus without transmembrane domains and signal peptides.Multiple sequences alignment showed that PatGTL1 contained two GT1 conserved domains,and was classified into GT-2 subfamily of Trihelix transcription factor family.According to the phylogenetic tree,PatGTL1 was clustered closely with Sesamum indicum SiGTL1.The qRT-PCR analysis indicated that PatGTL1 expressed in the root,stem,and leaf in patchouli,particularly with the highest expression in the young leaf.The PatGTL1 expression was significantly upregulated by MeJA and showed an increasing trend at 3—24 h after treatment,but downregulated by drought stress.The highest expression of PatGTL1 was showed at 3 h after salt treatment,and at 12 h after cold treatment.These results enrich the Trihelix transcription factor family study of patchouli.

Key words: Pogostemon cablin, Trihelix, GTL1, Gene cloning, Expression pattern, Subcellular localization

李俊仁, 吴带娣, 赵公政, 陈秀珍. 广藿香Trihelix转录因子家族基因PatGTL1的克隆、亚细胞定位及表达分析[J]. 华北农学报, 2024, 39(6): 84-93. doi: 10.7668/hbnxb.20195190.

LI Junren, WU Daidi, ZHAO Gongzheng, CHEN Xiuzhen. Cloning,Subcellular Localization and Expression Analysis of PatGTL1 Gene of Trihelix Family from Pogostemon cablin[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 84-93. doi: 10.7668/hbnxb.20195190.

http://www.hbnxb.net/CN/Y2024/V39/I6/84

图/表 9

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	SignaIP 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	SOPMA	https://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html
三级结构 Tertiary structure	SWISS-MODEL	https://swissmodel.expasy.org/

图1 广藿香PatGTL1基因的PCR扩增电泳条带

Fig.1 Agarose gel electrophoresis of PCR amplification for patchouli PatGTL1

图2 广藿香和其他植物GTL1的多序列比对

Fig.2 Amino acid sequence alignment of Trihelix transcription factor GTL1 in patchouli and other plant species

图3 PatGTL1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 PatGTL1

图4 PatGTL1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 PatGTL1

图5 PatGTL1的亚家族分类(A)及系统进化树分析(B)

Fig.5 The subfamily classification of PatGTL1(A)and the phylogenetic tree transcription factor GTL1 in patchouli and other plant species(B)

图6 PatGTL1蛋白亚细胞定位

Fig.6 Subcellular localization of PatGTL1 protein

图7 广藿香PatGTL1基因在不同组织中的相对表达量不同小写字母表示组织间差异显著(P<0.05)。图8同。

Fig.7 Expression profile of PatGTL1 in different tissues of patchouli The different lowercase letters indicate that the difference is significant between tissues at the 0.05 level.The same as Fig.8.

图8 广藿香PatGTL1基因在茉莉酸甲酯处理、盐胁迫、干旱胁迫及冷胁迫下的表达模式

Fig.8 Expression profiles of PatGTL1 under treatments of MeJA,salt,drought,and cold

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广藿香Trihelix转录因子家族基因PatGTL1的克隆、亚细胞定位及表达分析

Cloning,Subcellular Localization and Expression Analysis of PatGTL1 Gene of Trihelix Family from Pogostemon cablin

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广藿香Trihelix转录因子家族基因PatGTL1的克隆、亚细胞定位及表达分析

Cloning,Subcellular Localization and Expression Analysis of PatGTL1 Gene of Trihelix Family from Pogostemon cablin

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