Functional Analysis of the Transcription Factor MpR2R3-MYB17 in Regulating Gemma Development in Marchantia polymorpha L.

doi:10.13560/j.cnki.biotech.bull.1985.2025-0511

Abstract

Abstract:

Objective To investigate the functional role and molecular mechanisms of the MpR2R3-MYB17 gene in regulating gemma development in Marchantia polymorpha L., providing novel insights into the regulatory network of gemma formation. Method Based on the reference genome information of M. polymorpha L., the full-length CDS sequence of MpR2R3-MYB17 was cloned. Bioinformatics analysis of its encoded protein was performed, and transient transformation in tobacco was conducted to detect the subcellular localization of MpR2R3-MYB17. The overexpression vector and CRISPR/Cas9 editing vector were constructed, and the transformed strains were obtained by Agrobacterium-mediated non-tissue culture-dependent transformation method to assess phenotypic changes. Result Bioinformatics analysis showed that the open reading frame of the MpR2R3-MYB17 was 1 277 bp, encoding 421 amino acids, had two conserved SANT domains, and was an R2R3-type MYB transcription factor. Phylogenetic analysis revealed that MpR2R3-MYB17 and the ninth subfamily of Arabidopsis R2R3-MYB converged into one branch, and the relationship with AtMYB17 was the closest. Subcellular localization analysis confirmed that the MpR2R3-MYB17 transcription factor was localized in the nucleus, suggesting its potential direct involvement in transcriptional regulation. The overexpression of MpR2R3-MYB17 caused a significant increase in gemma cup density and gemma number compared with wild or development of undifferentiated cell clumps (highly expressed) on the thallus of M. polymorpha L. The density of the gemma cup on the thallus and the number of the gemma in each gemma cup in the edited individual significantly reduced compared to the wild. Conclusion MpR2R3-MYB17, as a nuclear localization protein, is involved in regulating both gemma cup and gemma development.

Key words: Marchantia polymorpha L., MpR2R3-MYB17, transcription factor, gemma development, subcellular localization, overexpression, CRISPR/Cas9, asexual reproduction

ZENG Ting, ZHANG Lan, LUO Rui. Functional Analysis of the Transcription Factor MpR2R3-MYB17 in Regulating Gemma Development in Marchantia polymorpha L.[J]. Biotechnology Bulletin, 2026, 42(1): 208-217.

Figures/Tables 9

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence （5′‒3′）	用途 Usage
M-F	GCTCTAGAATGGGGAGAGCGCCTTG	基因克隆
M-R	GCCCCCGGGTCAGCGCTCCATGACGGC	基因克隆
gRT1	TGCTGCGATAAGCAGGGGTTgttttagagctagaaat	sgRNA表达盒构建
gRT2	CGGGCACTTCCAAAACATGCgttttagagctagaaat
gRNA-R	CGGAGGAAAATTCCATCCAC
P-F	AGCGTGggtctcGCTCGACGCGTATCCATCCACTCCAAGCTC
P-R	TTCAGAggtctcTACCGACTAGTATGGAATCGGCAGCAAAGG
GUS-F	TCTGCGACGCTCACACCGAT	过表达阳性植株鉴定
GUS-R	GCCAACGCGCAATATGCCTT	过表达阳性植株鉴定
qMpR2R3-MYB17-F	CTACTGGAACACGCACCTGA	实时荧光定量PCR
qMpR2R3-MYB17-R	AGATGACTCAACTTGGAGCACA	实时荧光定量PCR
qMpAPT-F	CGAAAGCCCAAGAAGCTACC	内参基因
qMpAPT-R	GTACCCCCGGTTGCAATAAG	内参基因
Hyg-F	GTCGTCCATCACAGTTTGCC	突变体阳性鉴定
Hyg-R	TGCTTGACATTGGGGAGTTT	突变体阳性鉴定
PegMYB17-F	TATATTTGCCGTTGCTGCCG	测序片段克隆
PegMYB17-R	AGGGTTCAAAGCGCGATTCA	测序片段克隆

Fig. 1 Bioinformatics analysis and phylogenetic treeA: Hydrophilicity/hydrophobicity prediction of theMpR2R3-MYB17 protein amino acid sequence. B: Phylogenetic tree. C: Conserved domain analysis of the MpR2R3-MYB17 protein (Red: SANT domain. Purple: Low-complexity region). D: Analysis of cis-regulatory elements in the MpR2R3-MYB17 gene promoter region

Fig. 2 RNA extraction, cloning of MpR2R3-MYB17 gene, and recombinant plasmid mapsA: RNA extraction. B: MpR2R3-MYB17 gene cloning, the product length is 1 277 bp. C: pBI121-MpR2R3-MYB17 recombinant plasmid map (labelled gene). D: pEGCas9Pubi-MpR2R3-MYB17 recombinant plasmid (T1 and T2: sgRNA target sequences. gRNA scaffold: gRNA structural backbone)

Fig. 3 Subcellular localization of MpR2R3-MYB17 gene

Fig. 4 Screening of resistant plants, PCR identification of overexpressed plants and GUS stainingA: Marchantia polymorpha gemmae following Agrobacterium tumefaciens infection. B: Kanamycin-resistant plants selected after transformation. C: PCR confirmation of MpR2R3-MYB17-overexpressed transgenic lines. D: Histochemical GUS staining in MpR2R3-MYB17-overexpressed plants

Fig. 5 Phenotype of overexpressing plantsA: Wild type of M. polymorpha L.. B: Phenotypes of overexpressing plant lines (increased gemma cup density). C: Phenotypes of overexpressing plant lines (undifferentiated cell clumps). Undifferentiated cell clumps are indicated by red arrows. D: Enlarged view of undifferentiated cell clumps from panel C

Fig. 6 Phenotypic statistics and expression analysis of overexpressed plant linesA: Number of gemmae per gemma cup (gemmae/gemma cup). B: Gemma cup density (gemma cups per thallus). C: Relative expression of MpR2R3-MYB17 (wt: wild-type. MYB17-OE#1/2/3: Overexpressed lines with increased gemma cup density. MYB17-OE#4/5: Overexpressed lines producing undifferentiated cell clumps). Different lowercase letters indicate significant differences between groups (P<0.05)

Fig. 7 Mutant identification and sequence alignmentA: PCR identification of transgenic plants. B: Mutant sequence alignment (red box: 5′ non-coding region; purple boxes: exons; blue box: 3′ non-coding region. In the sequence alignment, red letters denote nucleotide substitutions. Dots "·": Base deletions. Horizontal line "—": Unmutated bases that are consistent with the reference sequence and are omitted)

Fig. 8 Phenotypic analysis of mutantsA: Wild type of M. polymorpha L.. B:Mutant lines. C:Number of gemmae per gemma cup (gemmae/gemma cup). D: Gemma cup density (gemma cups per thallus). myb17 indicates the phenotypic statistics of three independent mutant lines, myb17-1, myb17-2 and myb17-3, and different lowercase letters indicate significant differences between groups (P<0.01)

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