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

doi:10.7668/hbnxb.20195335

Abstract

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

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.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20195335

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

Figures/Tables 10

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/

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

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

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

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

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

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

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

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).

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).

References 42

[1]	国家药典委员会. 中华人民共和国药典(一部)[S]. 北京: 中国医药科技出版社, 2020.
	Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China.Part one[S]. Beijing: China Medical Science and Technology Press, 2020.
[2]	Swamy M K, Sinniah U R. Patchouli(Pogostemon cablin Benth.):botany,agrotechnology and biotechnological aspects[J]. Industrial Crops and Products, 2016, 87:161-176.doi:10.1016/j.indcrop.2016.04.032.
[3]	van Beek T A, Joulain D. The essential oil of patchouli,Pogostemon cablin:a review[J]. Flavour and Fragrance Journal, 2018, 33(1):6-51.doi:10.1002/ffj.3418.
[4]	Chen X Z, Li J R, Wang X B, Zhong L T, Tang Y, Zhou X X, Liu Y T, Zhan R T, Zheng H, Chen W W, Chen L K. Full-length transcriptome sequencing and methyl jasmonate-induced expression profile analysis of genes related to patchoulol biosynthesis and regulation in Pogostemon cablin[J]. BMC Plant Biology, 2019, 19(1):266.doi:10.1186/s12870-019-1884-x.
[5]	Chen X Z, Li J R, Liu Y T, Wu D D, Huang H L, Zhan R T, Chen W W, Chen L K. PatSWC4,a methyl jasmonate-responsive MYB(v-myb avian myeloblastosis viral oncogene homolog)-related transcription factor,positively regulates patchoulol biosynthesis in Pogostemon cablin[J]. Industrial Crops and Products, 2020, 154:112672.doi:10.1016/j.indcrop.2020.112672.
[6]	Chen X Z, Wang X B, Wu D D, Li J R, Huang H L, Wang X L, Zhan R T, Chen L K. PatDREB transcription factor activates patchoulol synthase gene promoter and positively regulates jasmonate-induced patchoulol biosynthesis[J]. Journal of Agricultural and Food Chemistry, 2022, 70(23):7188-7201.doi:10.1021/acs.jafc.2c01660. pmid: 35654756
[7]	Huang H L, Wu D D, Guo T Y, Zhang D H, Wang X L, Zhuang J X, Zou X, Gong L Z, Zhan R T, Chen L K. The PcbZIP44 transcription factor inhibits patchoulol synthase gene expression and negatively regulates patchoulol biosynthesis in Pogostemon cablin[J]. Industrial Crops and Products, 2022, 188:115561.doi:10.1016/j.indcrop.2022.115561.
[8]	Wang X B, Tang Y, Huang H L, Wu D D, Chen X Z, Li J R, Zheng H, Zhan R T, Chen L K. Functional analysis of Pogostemon cablin farnesyl pyrophosphate synthase gene and its binding transcription factor PcWRKY44 in regulating biosynthesis of patchouli alcohol[J]. Frontiers in Plant Science, 2022, 13:946629.doi:10.3389/fpls.2022.946629.
[9]	Kaplan-Levy R N, Brewer P B, Quon T, Smyth D R. The trihelix family of transcription factors-light,stress and development[J]. Trends in Plant Science, 2012, 17(3):163-171.doi:10.1016/j.tplants.2011.12.002. pmid: 22236699
[10]	Green P J, Kay S A, Chua N H. Sequence-specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcS-3A gene[J]. The EMBO Journal, 1987, 6(9):2543-2549.doi:10.1002/j.1460-2075.1987.tb02542.x.
[11]	向小雪, 娄红梅, 杨庆玲. Trihelix转录因子家族研究进展[J]. 安徽农业科学, 2022, 50(6):7-11. doi:10.3969/j.issn.0517-6611.2022.06.002.
	Xiang X X, Lou H M, Yang Q L. Research progress of trihelix transcription factor family[J]. Journal of Anhui Agricultural Sciences, 2022, 50(6):7-11.
[12]	卢惠君, 李子义, 贺子航, 王玉成, 及晓宇. 植物Trihelix转录因子响应非生物胁迫的研究进展[J]. 分子植物育种, 2019, 17(10):3241-3246.doi:10.13271/j.mpb.017.003241.
	Lu H J, Li Z Y, He Z H, Wang Y C, Ji X Y. Research progress on response of plant trihelix transcription factor to abiotic stress[J]. Molecular Plant Breeding, 2019, 17(10):3241-3246.
[13]	Breuer C, Kawamura A, Ichikawa T, Tominaga-Wada R, Wada T, Kondou Y, Muto S, Matsui M, Sugimoto K. The trihelix transcription factor GTL1 regulates ploidy-dependent cell growth in the Arabidopsis trichome[J]. The Plant Cell, 2009, 21(8):2307-2322.doi:10.1105/tpc.109.068387.
[14]	Shibata M, Breuer C, Kawamura A, Clark N M, Rymen B, Braidwood L, Morohashi K, Busch W, Benfey P N, Sozzani R, Sugimoto K. gtl1 and df1 regulate root hair growth through transcriptional repression of ROOT hair defective 6-like 4 in Arabidopsis[J]. Development, 2018, 145(3):dev159707.doi:10.1242/dev.159707.
[15]	Li P, Li Z X, Xie G N, Zhang J R. Trihelix transcription factor ZmThx20 is required for kernel development in maize[J]. International Journal of Molecular Sciences, 2021, 22(22):12137.doi:10.3390/ijms222212137.
[16]	Zhang Y, Yin H, Li D, Zhu W W, Li Q L. Functional analysis of BADH gene promoter from Suaeda liaotungensis K[J]. Plant Cell Reports, 2008, 27(3):585-592.doi:10.1007/s00299-007-0459-8. pmid: 17924116
[17]	Li J M, Zhang M H, Sun J, Mao X R, Wang J, Wang J G, Liu H L, Zheng H L, Zhen Z, Zhao H W, Zou D T. Genome-wide characterization and identification of trihelix transcription factor and expression profiling in response to abiotic stresses in rice(Oryza sativa L.)[J]. International Journal of Molecular Sciences, 2019, 20(2):251.doi:10.3390/ijms20020251.
[18]	Li K Y, Fan Y, Zhou G Y, Liu X J, Chen S S, Chang X C, Wu W Q, Duan L L, Yao M X, Wang R, Wang Z L, Yang M F, Ding Y Q, Ren M J, Fan Y, Zhang L Y. Genome-wide identification,phylogenetic analysis,and expression profiles of trihelix transcription factor family genes in quinoa(Chenopodium quinoa Willd.)under abiotic stress conditions[J]. BMC Genomics, 2022, 23(1):499.doi:10.1186/s12864-022-08726-y.
[19]	郑玲, 李梦丹. 甜瓜Trihelix转录因子的鉴定与生物信息学分析[J]. 江苏农业科学, 2022, 50(16):44-49.doi:10.15889/j.issn.1002-1302.2022.16.007.
	Zheng L, Li M D. Identification and bioinformatics analysis of Trihelix transcription factors in melon[J]. Jiangsu Agricultural Sciences, 2022, 50(16):44-49.
[20]	付梦婕. 番茄Trihelix基因家族生物信息学分析[J]. 分子植物育种, 2023, 21(5):1408-1417.doi:10.13271/j.mpb.021.001408.
	Fu M J. Bioinformatics analysis of Trihelix gene family in Solanum lycopersicum[J]. Molecular Plant Breeding, 2023, 21(5):1408-1417.
[21]	Hu J, Liu T, Huo H M, Liu S Z, Liu M M, Liu C, Zhao M Z, Wang K Y, Wang Y, Zhang M P. Genome-wide characterization,evolutionary analysis,and expression pattern analysis of the trihelix transcription factor family and gene expression analysis under MeJA treatment in Panax ginseng[J]. BMC Plant Biology, 2023, 23(1):376.doi:10.1186/s12870-023-04390-w.
[22]	Tong Y, Huang H, Wang Y H. Genome-wide analysis of the trihelix gene family and their response to cold stress in Dendrobium officinale[J]. Sustainability, 2021, 13(5):2826.doi:10.3390/su13052826.
[23]	Wang J, Ouyang Y W, Wei Y Z, Kou J J, Zhang X H, Zhang H N. Identification and characterization of trihelix transcription factors and expression changes during flower development in pineapple[J]. Horticulturae, 2022, 8(10):894.doi:10.3390/horticulturae8100894.
[24]	Zhao D B, Gao F J, Guan P Y, Gao J S, Guo Z H, Guo J J, Cui H N, Li Y J, Zhang G J, Li Z, Guo L H. Identification and analysis of differentially expressed trihelix genes in maize(Zea mays)under abiotic stresses[J]. PeerJ, 2023, 11:e15312.doi:10.7717/peerj.15312.
[25]	Zhao Y Y, Liang J C, Wang Z Q, Yan T X, Yan X W, Wei W L, Le M W, Sun J. Genome-wide identification and expression analysis of the trihelix transcription factor family in sesame(Sesamum indicum L.)under abiotic stress[J]. Molecular Biology Reports, 2023, 50(10):8281-8295.doi:10.1007/s11033-023-08640-w.
[26]	Lang Z L, Xu Z L, Li L Y, He Y Q, Zhao Y, Zhang C, Hong G J, Zhang X Y. Comprehensive genomic analysis of trihelix family in tea plant(Camellia sinensis)and their putative roles in osmotic stress[J]. Plants, 2024, 13(1):70.doi:10.3390/plants13010070.
[27]	Li J R, Chen X Z, Zhou X X, Huang H L, Wu D D, Shao J Q, Zhan R T, Chen L K. Identification of trihelix transcription factors in Pogostemon cablin reveals PatGT-1 negatively regulates patchoulol biosynthesis[J]. Industrial Crops and Products, 2021, 161:113182.doi:10.1016/j.indcrop.2020.113182.
[28]	Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts:a versatile cell system for transient gene expression analysis[J]. Nature Protocols, 2007, 2(7):1565-1572.doi:10.1038/nprot.2007.199.
[29]	向小雪. Trihelix转录因子SlASIL2在番茄生长发育及非生物胁迫中的功能研究[D]. 重庆: 重庆大学, 2022.doi:10.27670/d.cnki.gcqdu.2022.002510.
	Xiang X X. Functional study of trihelix transcription factor SlASIL2 in tomato growth and development and abiotic stresses[D]. Chongqing: Chongqing University, 2022.
[30]	Tian F, Yang D C, Meng Y Q, Jin J P, Gao G. PlantRegMap:charting functional regulatory maps in plants[J]. Nucleic Acids Research, 2020, 48(D1):D1104-D1113.doi:10.1093/nar/gkz1020.
[31]	Ruiz K A, Pelletier J M, Wang Y C, Feng M J, Behr J S, Ðào T Q, Li B H, Kliebenstein D, Harada J J, Jenik P D. A reevaluation of the role of the ASIL trihelix transcription factors as repressors of the seed maturation program[J]. Plant Direct, 2021, 5(10):e345.doi:10.1002/pld3.345.
[32]	Willmann M R, Mehalick A J, Packer R L, Jenik P D. MicroRNAs regulate the timing of embryo maturation in Arabidopsis[J]. Plant Physiology, 2011, 155(4):1871-1884.doi:10.1104/pp.110.171355. pmid: 21330492
[33]	García-Cano E, Magori S, Sun Q, Ding Z H, Lazarowitz S G, Citovsky V. Interaction of Arabidopsis trihelix-domain transcription factors VFP3 and VFP5 with Agrobacterium virulence protein VirF[J]. PLoS One, 2015, 10(11):e0142128.doi:10.1371/journal.pone.0142128.
[34]	Xu H Y, Shi X X, He L, Guo Y, Zang D D, Li H Y, Zhang W H, Wang Y C. Arabidopsis thaliana trihelix transcription factor AST1 mediates salt and osmotic stress tolerance by binding to a novel AGAG-box and some GT motifs[J]. Plant and Cell Physiology, 2018, 59(5):946-965.doi:10.1093/pcp/pcy032.
[35]	Luo J L, Tang S H, Mei F L, Peng X J, Li J, Li X F, Yan X H, Zeng X H, Liu F, Wu Y H, Wu G. BnSIP1-1,a trihelix family gene,mediates abiotic stress tolerance and ABA signaling in Brassica napus[J]. Frontiers in Plant Science, 2017, 8:44.doi:10.3389/fpls.2017.00044.
[36]	Koryachko A, Matthiadis A, Muhammad D, Foret J, Brady S M, Ducoste J J, Tuck J, Long T A, Williams C. Clustering and differential alignment algorithm:identification of early stage regulators in the Arabidopsis thaliana iron deficiency response[J]. PLoS One, 2015, 10(8):e0136591.doi:10.1371/journal.pone.0136591.
[37]	Lopes Cardoso M I, Meijer A H, Rueb S, Machado J Q, Memelink J, Hoge J H C. A promoter region that controls basal and elicitor-inducible expression levels of the NADPH:cytochrome P450 reductase gene(Cpr)from Catharanthus roseus binds nuclear factor GT-1[J]. Molecular and General Genetics, 1997, 256(6):674-681.doi:10.1007/pl00008617. pmid: 9435792
[38]	Liu H F, Zhang T T, Liu Y Q, Liu R X, Zhang H Y, Rui L, Wang D R, Li C Y, Zhang S, You C X, Zhang Z L, Wang X F. The trihelix transcription factor MdSIP1-2 interacts with MdNIR1 promoter to regulate nitrate utilization in apple[J]. Environmental and Experimental Botany, 2024, 220:105669.doi:10.1016/j.envexpbot.2024.105669.
[39]	Fan M Z, Herburger K, Jensen J K, Zemelis-Durfee S, Brandizzi F, Fry S C, Wilkerson C G. A trihelix family transcription factor is associated with key genes in mixed-linkage glucan accumulation[J]. Plant Physiology, 2018, 178(3):1207-1221.doi:10.1104/pp.18.00978. pmid: 30224432
[40]	Völz R, Kim S K, Mi J N, Mariappan K G, Guo X J, Bigeard J, Alejandro S, Pflieger D, Rayapuram N, Al-Babili S, Hirt H. The Trihelix transcription factor GT2-like 1(GTL1)promotes salicylic acid metabolism,and regulates bacterial-triggered immunity[J]. PLoS Genetics, 2018, 14(10):e1007708.doi:10.1371/journal.pgen.1007708.
[41]	Yang W Z, Hu J Y, Behera J R, Kilaru A, Yuan Y P, Zhai Y H, Xu Y F, Xie L H, Zhang Y L, Zhang Q Y, Niu L X. A tree peony trihelix transcription factor PrASIL1 represses seed oil accumulation[J]. Frontiers in Plant Science, 2021, 12:796181.doi:10.3389/fpls.2021.796181.
[42]	Zhu L, Hu J, Li R Q, Liu C, Jiang Y, Liu T, Liu M M, Zhao M Z, Wang Y, Wang K Y, Zhang M P. Transcriptome-wide integrated analysis of the PgGT25-04 gene in controlling ginsenoside biosynthesis in Panax ginseng[J]. Plants, 2023, 12(10):1980.doi:10.3390/plants12101980.

Please choose a citation manager

Content to export