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

doi:10.7668/hbnxb.20195190

Abstract

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

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.

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Figures/Tables 9

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/

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

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

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

Fig.4 Prediction of secondary structure(A)and tertiary structure(B)of PatGTL1

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

Fig.6 Subcellular localization of PatGTL1 protein

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.

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

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