转录因子MpR2R3-MYB17调控地钱胞芽发育的功能研究

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

摘要/Abstract

摘要：

目的探究MpR2R3-MYB17基因在地钱胞芽发育调控中的功能及分子机制，为进一步揭示地钱胞芽发育调控机制提供新的依据。方法基于地钱基因组信息克隆MpR2R3-MYB17的全长CDS序列，对其编码蛋白质进行生物信息学分析并瞬时转化烟草检测MpR2R3-MYB17亚细胞定位。构建过表达载体和CRISPR/Cas9基因编辑载体，利用农杆菌介导的非组培依赖的地钱转化方法获得MpR2R3-MYB17过表达株系和突变体株系以评估表型变化。结果生物信息学分析表明，MpR2R3-MYB17的开放阅读框的长度为1 277 bp，编码421个氨基酸，具有2个保守的SANT序列，是R2R3型MYB转录因子。系统发育树揭示MpR2R3-MYB17与拟南芥R2R3-MYB第9亚家族聚为一支，其中与AtMYB17同源性最高。亚细胞定位分析表明，MpR2R3-MYB17蛋白定位于细胞核内，提示其可能直接参与转录调控过程。表型分析表明，与野生型相比，过表达株系的叶状体上胞芽杯密度和胞芽数量显著增加，或在高表达状态下形成未分化细胞团，且叶状体的生长受到抑制。突变体株系的胞芽杯密度及单个胞芽杯内胞芽数量均较野生型显著降低。结论 MpR2R3-MYB17作为细胞核定位的转录因子参与调控地钱胞芽杯和胞芽的发育过程。

关键词: 地钱, MpR2R3-MYB17, 转录因子, 胞芽发育, 亚细胞定位, 过表达, CRISPR/Cas9, 无性繁殖

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

曾厅, 张兰, 罗睿. 转录因子MpR2R3-MYB17调控地钱胞芽发育的功能研究[J]. 生物技术通报, 2026, 42(1): 208-217.

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.

图/表 9

表1 本研究使用的引物

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

图1 生物信息学分析和系统发育树A：MpR2R3-MYB17蛋白氨基酸序列亲疏水性预测结果；B：系统发育树；C：MpR2R3-MYB17蛋白保守结构域分析（红色部分：SANT结构域；紫色部分：低复杂度区域）；D：MpR2R3-MYB17启动子区顺式作用元件分析

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

图2 RNA提取、MpR2R3-MYB17基因克隆、重组质粒图A：RNA提取结果；B：MpR2R3-MYB17基因克隆，产物长度为1 277 bp；C：pBI121-MpR2R3-MYB17重组质粒图（GUS：标记基因）；D：pEGCas9Pubi-MpR2R3-MYB17重组质粒（T1、T2为sgRNA靶点序列；gRNA scaffold为gRNA骨架区）

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)

图3 MpR2R3-MYB17基因亚细胞定位

Fig. 3 Subcellular localization of MpR2R3-MYB17 gene

图4 抗性植株筛选、过表达植株PCR鉴定和GUS染色A：农杆菌侵染处理后的地钱胞芽；B：经Kana抗性筛选获得的抗性植株；C：MpR2R3-MYB17过表达转基因植株的PCR阳性鉴定；D：MpR2R3-MYB17过表达植株的组织化学GUS染色

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

图5 过表达植株表型A：地钱野生型植株；B：MpR2R3-MYB17过表达株系表型（胞芽杯密度增加）；C：MpR2R3-MYB17过表达株系表型（产生未分化细胞团），红色箭头表示未分化细胞团；D：图C中未分化细胞团的放大图

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

图6 过表达株系表型统计与表达分析A：单个胞芽杯内胞芽数量（个/胞芽杯）；B：胞芽杯密度（个/叶状体）；C：MpR2R3-MYB17基因相对表达量（wt：野生型；MYB17-OE#1/2/3：胞芽杯数量增多的过表达株系；MYB17-OE#4/5：产生未分化细胞团的过表达株系）；不同小写字母表示组间差异显著（P<0.05）

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)

图7 突变体鉴定与序列比对A：转基因植株PCR鉴定；B：突变体序列比对（红色方框：5′非编码区；紫色方框：外显子区域；黑色横线：内含子区域；蓝色方框：3′非编码区；序列比对中红色字母表示碱基替换；圆点“·”表示碱基缺失；横线“—”表示与参考序列一致而被省略的未突变碱基）

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)

图8 突变体表型分析A：野生型植株；B：突变体株系；C：单个胞芽杯内胞芽数量（个/胞芽杯）；D：胞芽杯密度（个/叶状体）。myb17代表3个独立突变体株系myb17-1、myb17-2、myb17-3的表型统计结果，不同小写字母表示组间差异显著（P<0.01）

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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