Cloning and Functional Analysis of <i>WINDs</i> gene in <i>Populus euphratica</i>

doi:10.13523/j.cb.2205005

China Biotechnology

2022, Vol. 42

Issue (10): 9-20 DOI: 10.13523/j.cb.2205005

Cloning and Functional Analysis of WINDs gene in Populus euphratica

Si-jia HOU,Jing CHEN,Jian-qiao MENG,Jun-hong DU,Cong WANG,Dan LIANG,Rong-ling WU,Yun-qian GUO^**(

)

College of Biological Science and Technology, Center for Computational Biology, National Engineering Laboratory for Tree Breeding, Beijing Forestry University, Beijing 100083, China

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Abstract

Objective: WIND (WOUND INDUCED DEDIFFERENTIATION) are important transcription factors belonging to ERF/AP2 (ETHYLENE RESPONSE FACTOR/ APETALA 2) family. The genes were first found to bind to ethylene response element GCC-BOX and dehydration response element DRE in Arabidopsis thaliana to respond to drought signals and regulate ethylene levels. Recent studies have found that WIND genes also play a key role in plant wound signal response, callus formation and adventitious shoot production. Previous studies have explored the mechanisms of the WIND gene controlling callus formation and adventitious shoot regeneration in Arabidopsis thaliana. However, the functions of WIND in woody plants remain unclear. The functions of WIND genes in wound signal response and adventitious shoot regeneration will be explored in Populus euphratica. A theoretical basis for solving the regeneration problem of P. euphratica at the molecular level will be provided. Methods: Gene cloning, qRT-PCR and transgenic phenotype analysis were used. Results: Two WIND genes of P.euphratica were cloned as PeWIND1 and PeWIND2, respectively. The coding regions were 1 050 bp and 1 032 bp, encoding 349 and 343 amino acids, respectively. Subcellular localization analysis showed that both genes were functional in the nucleus. Gene expression analysis showed that PeWIND1 and PeWIND2 expressed in roots, stems, leaves, and calli. The highest expression level was found in calli. The time course expression analysis showed that the expression levels of PeWIND1 and PeWIND2 were first increased and then decreased within 24 h after wound stimulation, and peaked at 1 h after wound stimulation. Phenotypic statistics of transgenic plants showed that the regeneration ability of adventitious shoots was enhanced after overexpression of PeWIND1 and PeWIND2. Conclusion: PeWIND1 and PeWIND2 responded to wound signal and the expression levels of PeWIND1 and PeWIND2 were first increased and then decreased within 24 h after wound stimulation. PeWIND1 and PeWIND2 promoted adventitious shoot regeneration from stems of poplars.

Key words： Plant regeneration Populus euphratica PeWIND Wound response

Received: 04 May 2022 Published: 04 November 2022

ZTFLH:

Q812

Corresponding Authors: Yun-qian GUO E-mail: guoyunqian@bjfu.edu.cn

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	Si-jia HOU
	Jing CHEN
	Jian-qiao MENG
	Jun-hong DU
	Cong WANG
	Dan LIANG
	Rong-ling WU
	Yun-qian GUO

Cite this article:

Si-jia HOU,Jing CHEN,Jian-qiao MENG,Jun-hong DU,Cong WANG,Dan LIANG,Rong-ling WU,Yun-qian GUO. Cloning and Functional Analysis of WINDs gene in Populus euphratica. China Biotechnology, 2022, 42(10): 9-20.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2205005 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I10/9

Table 1 Primer sequences used in PCR for amplifying target genes

Table 2 Primer sequences with BamHI and SalI restriction sites used in PCR for amplifying target genes

Table 3 Primer sequences used in qRT-PCR

Fig.1 Electropherograms of PeWIND1, PeWIND2 gene clones M:Marker

Fig.2 PeWIND gene maps (a) Comparison of PeWIND1 and NCBI prediction results (b)Comparison of PeWIND2 and NCBI prediction results

Fig.3 Comparison of amino acid sequence alignment of PeWIND with PtrRAP2-4、PalRAP2-13-like、PdeKAH8508800.1 and PtoKAG6774166.1

Fig.4 Protein domain prediction of PeWIND1, PeWIND2 (a) PeWIND1 (b) PeWIND2 AP2:AP2 superfamily

Fig.5 Three-dimensional structure prediction of PeWIND1, PeWIND2 proteins (a) PeWIND1 (b) PeWIND2

Fig.6 Phylogenetic analysis of PeWIND1, PeWIND2 proteins from different plant species In this paper, PeuRAP2-4-like was named PeWIND1, PeuRAP2-13 was named PeWIND2;Ach: Actinidia chinensis var. chinensis;Apr: Abrus precatorius;Ccl: Citrus clementina;Cil: Carya illinoinensis;Cpa: Carica papaya;Dzi: Durio zibethinus;Ghi: Gossypium hirsutum;Gar: Gossypium arboreum;Hbr: Hevea brasiliensis;Hum: Herrania umbratical;Jcu: Jatropha curcas;Jre: Juglans regia;Mes: Manihot esculenta;Mno: Morus notabilis;Mru: Morella rubra;Min: Mangifera indica;Pra: Prosopis alba;Pal: Populus alba;Peu: Populus euphratica;Pmu: Prunus mume;Ptr: Populus trichocarpa;Pde: Populus deltoides; Pto: Populus tomentosa; Pve: Pistacia vera;Qlo: Quercus lobata;Qsu: Quercus suber;Rco: Ricinus communis;Sto: Senna tora; Ssu: Spatholobus suberectus;Twi: Tripterygium wilfordii;Tca: Theobroma cacao;Tsp: Telopea speciosissim;Vra: Vigna radiata var. radiata;Vvi: Vitis vinifera;Vri: Vitis riparia

Fig.7 Laser confocal microscopy of transgenic Nicotiana benthamiana leaves Red is chloroplast autofluorescence, green is GFP signal. Scale bar, 25 μm

Fig.8 Tissue expression specificity of PeWIND gene in Populus euphratica was detected by qRT-PCR (a) PeWIND1 (b) PeWIND2 The figure shows the relative expression levels of PeWIND gene in Populus euphratica roots, stems, leaves and callus explants respectively (the relative expression levels are obtained by comparing PeWIND gene expression levels in roots)

Fig.9 Time expression specificity of PeWIND gene in Populus euphratica was detected by qRT-PCR (a)PeWIND1 (b)PeWIND2 The figure shows the relative expression levels of PeWIND1, PeWIND2 genes at 0, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h and 24 h after wound treatment in Populus euphratica leaf explants (the relative expression levels are obtained by comparing the expression levels of each gene at 0)

Fig.10 Relative expression level of PeWIND gene in Populus euphratica was detected by qRT-PCR (a)PeWIND1 (b)PeWIND2 WT:wild type; W1-C: 35S∷PeWIND1-C strain; W1-21: 35S∷PeWIND1-21 strain; W2-26: 35S∷PeWIND2-26 strain; EW2-31: 35S∷ PeWIND2-31 strain

Fig.11 WIND promotes shoot regeneration in vitro (a)Adventitious shoot regeneration from stem explants of wild type, W1-C, W1-21, W2-26 and W2-31. Stem explants were cultured on SIM for 20 days. Representative images of stem explants cultured on SIM for 20 days. Bar = 5 mm (b)Quantitative analysis of regeneration phenotype of adventitious shoots. The regeneration phenotype was determined by the number of regeneration shoots per explant. There were 15 explants per genotype and three groups of biological replicates (n = 45 for each genotype). The T test was used to determine whether the difference was statistically significant (^*** P<0.001)


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