Characterization of the Activation Domain of SlMYB80 in Tomato and Its Function Validation during Pollen Development in Arabidopsis

doi:10.13560/j.cnki.biotech.bull.1985.2025-0354

Abstract

Abstract:

Objective MYB80 is a key regulatory factor in pollen wall formation in Arabidopsis and rice (Oryza sativa). To explore the function of the tomato homolog SlMYB80 transcription factor and identify its transcriptional activation domain may provide a theoretical basis for further enriching the genetic resources of MYB transcription factors in tomato male sterile lines. Method Using the cultivated tomato ‘Moneymaker’ as the wild-type material, the SlMYB80 gene was cloned from the cDNA of its flower buds. The cauliflower mosaic virus CaMV 35s promoter was employed to drive the fusion of the SlMYB80 coding region (CDS) with the enhanced green fluorescent protein (eGFP) coding sequence, which was used to detect the subcellular localization of the SlMYB80 protein. Moreover, the coding region of the gene was divided into four fragments, which were separately ligated into the yeast expression vectors pGTBKT7 and co-transformed with pGTADT7 into yeast respectively for interaction assays to identify its transcriptional activation domain. Additionally, the binary vector containing SlMYB80CDS driven by the Male Sterile 188 (MS188)/AtMYB80 promoter was constructed and introduced into ms188 heterozygotes mutant and ms188 homozygote transgenic lines were obtained. The biological function of SlMYB80 was investigated by this genetic complementation. Result The amino acid sequence alignment and phylogenetic tree analysis of MYB80 indicated that the MYB80 amino acid sequence is highly conserved in land plants, particularly in the R2R3 DNA-binding domain region. Subcellular localization experiments in tobacco demonstrated that SlMYB80-eGFP is localized in the nucleus. Yeast interaction assays revealed that the transcriptional activation domain of SlMYB80 is located at the C-terminal 17 amino acid residues of the peptide chain. The complementation results indicated that the expressions of the SlMYB80 in the flower buds of transgenic complemented ms188 mutant plants were enabled to produce a few normal pollen grains. Conclusion SlMYB80, as the orthologous gene of MS188/AtMYB80 in Arabidopsis, encodes an R2R3 MYB transcription factor localized in the nucleus. The 17 amino acids at the C-terminus of its polypeptide chain constitute an activation domain. The expression of SlMYB80 in ms188 partially complements the pollen abortion phenotype of the ms188 mutant, revealing the functional conservation of MYB80 transcription factors.

Key words: tomato, SlMYB80, transcription factor, transcriptional activation domain, male sterility, homology analysis, yeast hybrid, functional complement

ZHANG Yu-qing, DONG Li-xue, ZHANG Bao-yue, ZHANG Ying, LIU Xue-ao, XIONG Shuang-xi, ZHANG Hong-xia. Characterization of the Activation Domain of SlMYB80 in Tomato and Its Function Validation during Pollen Development in Arabidopsis[J]. Biotechnology Bulletin, 2025, 41(10): 222-232.

Figures/Tables 7

Table 1 Primers sequences used in this article

引物名称 Primer name	引物序列 Primer sequence （5′-3′）	引物用途 Primer usage
β-tub qRT F	GATTTCAAAGATTAGGGAAGAGTA	RT-qPCR
β-tub qRT R	GTTCTGAAGCAAATGTCATAGAG
SlMYB80 qRT F	ATTATCTGGTATGGGAATTGATCC
SlMYB80 qRT R	TTAACATGACTTGTACTTGGTGCA
SlMYB80fullCDS-BK F	catatggccatggaggccATGGGAAGAATTCCATGTTGTG	酵母杂交 Yeast hybrid
SlMYB80fullCDS-BK R	aggtcgacggatccccggTCAAACCATTGGATTCATTAGAT
SlMYB80fullCDS-AD F	atggccatggaggccagtATGGGAAGAATTCCATGTTGTG
SlMYB80fullCDS-AD R	tcgatgcccacccgggtgTCAAACCATTGGATTCATTAGAT
SlMYB80CDS（319 aa）-BK F	catatggccatggaggccATGGGAAGAATTCCATGTTGTG
SlMYB80CDS（319 aa）-BK R	aggtcgacggatccccggTCAAGTATTGAACATAGTTGTTGATCC
SlMYB80CDS（298 aa）-BK F	catatggccatggaggccATGGGAAGAATTCCATGTTGTG
SlMYB80CDS（298 aa）-BK R	aggtcgacggatccccggTCAACAATTATCATTGCTAATTTTC
SlMYB80CDS（272 aa）-BK F	catatggccatggaggccATGGGAAGAATTCCATGTTGTG
SlMYB80CDS（272 aa）-BK R	aggtcgacggatccccggTCATGCTGTGCATGTACTTCCA
SlMYB80CDS-35S-GFP F	gagctcggtacccggATGGGAAGAATTCCATGTTGTGA	亚细胞定位 Subcellular localization
SlMYB80CDS-35S-GFP R	catgtcgactctagaAACCATTGGATTCATTAGATCATC
ProAtMS188 F	ttcgagctcggtacccggAAGTTGTGTTTTTTCCCAAGTCA	互补验证 Complementary verification
ProAtMS188 R	caccattctagaggatccTTCTTCTTTCTTTCTTTCTAGTTTTT
SlMYB80 full CDS F	gaaagaaagaaagaagaaATGGGAAGAATTCCATGTTGTGA
SlMYB80 full CDS R	cttgctcaccattctagaTCAAACCATTGGATTCATTAGATCATC
pAtMS188-SlMYB80 JD F	ATCTGAATGTAAAAGGAGTTGACC	转基因阳性植株鉴定 Transgenic positive plants identification
pAtMS188-SlMYB80 JD R	ATGTTGAGCAATGTAAGATGAAAG

Fig. 1 Phylogenetic analysis of SlMYB80 with proteins from other speciesMonocot: Monocotyledon. Dicot: Dicotyledon. Red point indicates SlMYB80 from tomato

Fig. 2 PCR amplification of the full-length coding sequence (CDS) of SlMYB80 (A) and identification of positive clones by colony PCR assay (B)In Fig. A, 1: the full-length SlMYB80 CDS amplified from cDNA template; 2: negative control (ddH2O). In Fig. B, 1, 2: two single colonies of Escherichia coli; 3: positive control; 4: negative control (ddH2O). M: DL 5 000 marker

Fig. 3 SlMYB80-GFP protein specifically localized on the nucleus of tobacco mesophyll cellsA: Schematic diagram of subcellular localization construct. B‒E: Confocal microscopy images of p35s:GFP pCAMBIA1300 empty vector Agrobacterium-infiltrated tobacco leaves. F‒I: Confocal microscopy images of p35s:SlMYB80CDS-GFP pCAMBIA1300 Agrobacterium-infiltrated tobacco leaves. B and F: GFP signal channel. C and G: mCherry signal channel. D and H: Overlay signal channel images. E and I: Bright field channel images. Bar = 20 μm

Fig. 4 PCR amplification of diverse fragments of SlMYB80 CDS in tomato and colony PCR screening of positive clonesA: PCR amplification of region 1 (amino acids 1‒272) DNA fragment. B: PCR amplification of region 2 (amino acids 1‒298) DNA fragment. C: PCR amplification of region 3 (amino acids 1‒319) DNA fragment. D: PCR amplification of region 4 (amino acids 1‒336) DNA fragment. E‒H: positive clones detected by colony PCR of E. coli with different segments ligated into the pGBKT7 vector. In Fig. A‒D: M: DL 5 000 marker; 1: PCR amplification of different regions of the SlMYB80CDS DNA fragment; 2: negative control (ddH2O). In Fig. E‒H: M: DL 5 000 marker; 1‒2: two single colonies of E. coli; 3: positive control; 4: negative control (ddH2O)

Fig. 5 Activation domain of SlMYB80 on the 17 amino acid residues at the C-terminus of the polypeptide chainA: Schematic diagram of the connection of diverse fragments of SlMYB80 to the vectors pGBKT7 and empty pGADT7. B: Alignment of amino acid sequences between MS188/AtMYB80 and SlMYB80 (The red line indicates the R2R3 DNA-binding domain, and the red box indicates the activation domain). C: Identification of the activation of yeast reporter gene expression by diverse fragments of SlMYB80 on SD media lacking different amino acids. -Trp/Leu: SD medium without Trp and Leu; -Ade/-His/-Trp/-Leu: SD medium without Ade, His, Trp and Leu; -Ade/-His/-Trp/-Leu/X-α-gal: SD medium added X-α-gal without Ade, His, Trp and Leu

Fig. 6 Full-length coding sequence (CDS) of SlMYB80 can partially complement the phenotype of the Arabidopsis ms188A: Col-0 wild-type (WT). B: ms188 mutant. C: pAtMYB80:SlMYB80CDS p2300 complementation of the ms188; bar=2 cm; the white arrow indicates the recovered siliques. D‒F: Alexander staining (D: wild-type anther; E: ms188 anther; F: anther of the complemented plants (the white arrow indicates the recovered pollen grains); bar=100 μm). G: PCR identification of transgenic plants (DL2000 marker; 1‒5: independent transgenic lines; 6: positive control; 7: negative control (WT); 8: negative control (ddH2O)). H: RT-qPCR analysis of the relative expressions of SlMYB80 in transgenic plants. t-student test, *** indicates P<0.01

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