番茄SlMYB80转录激活结构域的鉴定及其在拟南芥花粉发育中的功能验证

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

摘要/Abstract

摘要：

目的 MYB80是拟南芥和水稻中的一个花粉壁形成关键调节因子，探究番茄中同源基因SlMYB80的功能并鉴定其转录激活结构域，为进一步丰富MYB转录因子对番茄雄性不育系基因资源提供理论依据。方法以栽培番茄‘Moneymaker’为野生型材料，以其花苞cDNA为模板克隆SlMYB80基因。利用花椰菜花叶病毒CaMV 35s启动子驱动SlMYB80编码区（CDS）与绿色荧光蛋白eGFP编码序列融合，用于检测SlMYB80蛋白亚细胞定位。同时，将该基因编码区分成4个片段，分别连接酵母表达载体pGTBKT7，与pGTADT7共转酵母进行互作试验以确定其转录激活结构域。此外，构建拟南芥MS188（Male Sterile 188）/AtMYB80启动子驱动SlMYB80 CDS的融合双元载体pAtMYB80：SlMYB80并互补拟南芥ms188突变体，探究SlMYB80的生物学功能。结果 MYB80氨基酸序列比对和进化树结果表明，MYB80的氨基酸序列在陆生植物中十分保守，特别是R2R3 DNA结合结构域区域；烟草亚细胞定位试验表明，SlMYB80-eGFP定位于细胞核。酵母互作试验表明，SlMYB80转录激活结构域位于肽链C末端17个氨基酸残基；拟南芥ms188突变体互补结果表明，在转基因互补突变体植株的花苞中表达SlMYB80基因，能使部分花粉正常形成，从而恢复部分育性。结论 SlMYB80作为拟南芥MS188/AtMYB80的直系同源基因编码一个R2R3 MYB转录因子定位于细胞核中，其多肽链C末端17个氨基酸为激活结构域，能部分互补ms188突变体花粉败育的表型，揭示了MYB80转录因子功能的保守性。

关键词: 番茄, SlMYB80, 转录因子, 转录激活结构域, 雄性不育, 同源性分析, 酵母杂交, 功能互补

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

张雨晴, 董丽雪, 张宝月, 张颖, 刘雪敖, 熊双喜, 张洪霞. 番茄SlMYB80转录激活结构域的鉴定及其在拟南芥花粉发育中的功能验证[J]. 生物技术通报, 2025, 41(10): 222-232.

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.

图/表 7

表1 本文中使用的引物序列

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

图1 SlMYB80与其他物种蛋白的系统进化分析Monocot：单子叶植物； Dicot：双子叶植物；红点代表番茄SlMYB80

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

图2 PCR扩增SlMYB80全长编码区（CDS）（A）与菌落PCR检测阳性克隆（B）图A中，1：cDNA模板扩增出的SlMYB80 CDS全长；2：阴性对照（双蒸水）；图B中，1、2：大肠杆菌单克隆；3：阳性对照；4：阴性对照（双蒸水）；M：DL 5 000 marker

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

图3 SlMYB80-GFP蛋白定位于烟草叶肉细胞核中A：亚细胞定位构建示意图；B‒E：p35s：GFP pCAMBIA1300空载农杆菌注射烟草叶片激光共聚焦扫描显微镜拍摄照片；F‒I：p35s：SlMYB80CDS-eGFP pCAMBIA1300农杆菌注射烟草叶片激光共聚焦扫描显微镜拍摄照片；B和F：GFP信号通道；C和G：mCherry信号通道；D和H：叠加信号通道照片；E和I：明场通道照片；比例尺 = 20 μm

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

图4 PCR扩增番茄SlMYB80编码区（CDS）的不同区段和菌落PCR检测阳性克隆A：1区（SlMYB80CDS第1‒272个氨基酸）DNA片段PCR扩增；B：2区（SlMYB80CDS第1‒298个氨基酸）DNA片段PCR扩增；C：3区（SlMYB80CDS第1‒319个氨基酸）DNA片段PCR扩增；D：4区（SlMYB80CDS第1‒336个氨基酸）DNA片段PCR扩增；E‒H：不同区段连接pGBKT7载体大肠杆菌菌落PCR检测阳性克隆；图A‒D中，M：DL 5 000 marker；1：扩增SlMYB80CDS不同区段DNA片段；2：阴性对照（双蒸水）；图E‒H中，M：DL 5 000 marker；1‒2：大肠杆菌单克隆；3：阳性对照；4：阴性对照（双蒸水）

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)

图5 SlMYB80激活结构域位于多肽链C末端17个氨基酸残基上A：SlMYB80不同区段DNA片段连接pGBKT7和pGADT7空载体的示意图；B：拟南芥MS188/AtMYB80氨基酸序列对比图（红线代表R2R3 DNA结合结构域，红方框代表激活结构域）；C：不同缺氨基酸SD培养基上鉴定SlMYB80不同区段激活酵母报告基因表达情况；-Trp/-Leu：SD培养基缺少Trp和Leu；-Ade/-His/-Trp/-Leu：SD培养基缺少Ade、His、Trp和Leu；-Ade/-His/-Trp/-Leu/X-α-gal：SD培养基缺少Ade、His、Trp和Leu并添加X-α-gal

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

图6 SlMYB80全长编码区（CDS）可以部分互补拟南芥ms188突变体表型A：Col-0野生型（WT）拟南芥；B：ms188突变体拟南芥；C：pAtMYB80：SlMYB80CDS p2300互补ms188突变体，比例尺=2 cm，白色箭头代表恢复的角果；D‒F：亚历山大染色（D：野生型花药；E：ms188突变体花药；F：互补植株花药，白色箭头代表恢复的花粉；比例尺=100 μm）；G：PCR鉴定转基因植株（M：DL2000 marker；1‒5：转基因植株；6：阳性对照；7：阴性对照（WT）；8：阴性对照（ddH2O））；H：RT-qPCR检测转基因植株中SlMYB80相对表达量；t-student检验，***代表P<0.01

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

参考文献 37

[1]	李君明, 项朝阳, 王孝宣, 等. “十三五” 我国番茄产业现状及展望 [J]. 中国蔬菜, 2021(2): 13-20.
	Li JM, Xiang CY, Wang XX, et al. Current situation of tomato industry in China during ‘the thirteenth Five-Year Plan’ period and future prospect [J]. China Veg, 2021(2): 13-20.
[2]	胡雅丹, 伍国强, 刘晨, 等. MYB转录因子在调控植物响应逆境胁迫中的作用 [J]. 生物技术通报, 2024, 40(6): 5-22.
	Hu YD, Wu GQ, Liu C, et al. Roles of MYB transcription factor in regulating the responses of plants to stress [J]. Biotechnol Bull, 2024, 40(6): 5-22.
[3]	李翔, 李涛, 宫超, 等. 番茄雄性不育研究现状与展望 [J]. 广东农业科学, 2023, 50(2): 49-58.
	Li X, Li T, Gong C, et al. Current situation and prospect of research on male sterility of tomato [J]. Guangdong Agric Sci, 2023, 50(2): 49-58.
[4]	Patikoglou G, Burley SK. Eukaryotic transcription factor-DNA complexes [J]. Annu Rev Biophys Biomol Struct, 1997, 26: 289-325.
[5]	Prouse MB, Campbell MM. The interaction between MYB proteins and their target DNA binding sites [J]. Biochim Biophys Acta Gene Regul Mech, 2012, 1819(1): 67-77.
[6]	Zhang ZB, Zhu J, Gao JF, et al. Transcription factor AtMYB103 is required for anther development by regulating tapetum development, callose dissolution and exine formation in Arabidopsis [J]. Plant J, 2007, 52(3): 528-538.
[7]	Xiong SX, Lu JY, Lou Y, et al. The transcription factors MS188 and AMS form a complex to activate the expression of CYP703A2 for sporopollenin biosynthesis in Arabidopsis thaliana [J]. Plant J, 2016, 88(6): 936-946.
[8]	Wang K, Guo ZL, Zhou WT, et al. The regulation of sporopollenin biosynthesis genes for rapid pollen wall formation [J]. Plant Physiol, 2018, 178(1): 283-294.
[9]	Han Y, Zhou SD, Fan JJ, et al. OsMS188 is a key regulator of tapetum development and sporopollenin synthesis in rice [J]. Rice, 2021, 14(1): 4.
[10]	He Y, Wang J, Hu JG, et al. Knockdown of SlEMS1 causes male sterility in tomato [J]. Hortic Plant J, 2025, 11(2): 939-942.
[11]	Jeong HJ, Kang JH, Zhao MA, et al. Tomato male sterile 1035 is essential for pollen development and meiosis in anthers [J]. J Exp Bot, 2014, 65(22): 6693-6709.
[12]	Sun ZL, Cheng BH, Zhang YH, et al. SlTDF1: a key regulator of tapetum degradation and pollen development in tomato [J]. Plant Sci, 2025, 351: 112321.
[13]	Bao HH, Ding YM, Yang F, et al. Gene silencing, knockout and over-expression of a transcription factor ABORTED MICROSPORES (SlAMS) strongly affects pollen viability in tomato (Solanum lycopersicum) [J]. BMC Genomics, 2022, 23(): 346.
[14]	Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11 [J]. Mol Biol Evol, 2021, 38(7): 3022-3027.
[15]	Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees [J]. Mol Biol Evol, 1987, 4(4): 406-425.
[16]	He SC, Ma R, Liu ZJ, et al. Overexpression of BnaAGL11, a MADS-box transcription factor, regulates leaf morphogenesis and senescence in Brassica napus [J]. J Agric Food Chem, 2022, 70(11): 3420-3434.
[17]	Li ZJ, Peng RH, Tian YS, et al. Genome-wide identification and analysis of the MYB transcription factor superfamily in Solanum lycopersicum [J]. Plant Cell Physiol, 2016, 57(8): 1657-1677.
[18]	杨晶婷, 余艳, 高晓蓉, 等. MYB类转录因子调控植物耐逆机制的研究进展 [J]. 杭州师范大学学报: 自然科学版, 2021, 20(6): 621-627.
	Yang JT, Yu Y, Gao XR, et al. Advances in plant MYB transcription factors regulation mechanisms to various stresses [J]. J Hangzhou Norm Univ Nat Sci Ed, 2021, 20(6): 621-627.
[19]	Fang Q, Wang Q, Mao H, et al. AtDIV2, an R-R-type MYB transcription factor of Arabidopsis, negatively regulates salt stress by modulating ABA signaling [J]. Plant Cell Rep, 2018, 37(11): 1499-1511.
[20]	Zhang X, Chen LC, Shi QH, et al. SlMYB102, an R2R3-type MYB gene, confers salt tolerance in transgenic tomato [J]. Plant Sci, 2020, 291: 110356.
[21]	Wang ML, Hao J, Chen XH, et al. SlMYB102 expression enhances low-temperature stress resistance in tomato plants [J]. PeerJ, 2020, 8: e10059.
[22]	Liu XJ, Ban Z, Yang YY, et al. The yellowhorn MYB transcription factor MYB30 is required for wax accumulation and drought tolerance [J]. Tree Physiol, 2024, 44(10): tpae111.
[23]	Zhang Y, Zhang B, Yang TW, et al. The GAMYB-like gene SlMYB33 mediates flowering and pollen development in tomato [J]. Hortic Res, 2020, 7: 133.
[24]	Stevens VA, Murray BG. Studies on heteromorphic self-incompatibility systems: the cytochemistry and ultrastructure of the tapetum of Primula obconica [J]. J Cell Sci, 1981, 50: 419-431.
[25]	Vizcay-Barrena G, Wilson ZA. Altered tapetal PCD and pollen wall development in the Arabidopsis ms1 mutant [J]. J Exp Bot, 2006, 57(11): 2709-2717.
[26]	Parish RW, Li SF. Death of a tapetum: a programme of developmental altruism [J]. Plant Sci, 2010, 178(2): 73-89.
[27]	Fu ZZ, Yu J, Cheng XW, et al. The rice basic helix-loop-helix transcription factor TDR INTERACTING PROTEIN2 is a central switch in early anther development [J]. Plant Cell, 2014, 26(4): 1512-1524.
[28]	Wang N, Li X, Zhu J, et al. Molecular and cellular mechanisms of photoperiod- and thermo-sensitive genic male sterility in plants [J]. Mol Plant, 2025, 18(1): 26-41.
[29]	Gu JN, Zhu J, Yu Y, et al. DYT1 directly regulates the expression of TDF1 for tapetum development and pollen wall formation in Arabidopsis [J]. Plant J, 2014, 80(6): 1005-1013.
[30]	Lou Y, Xu XF, Zhu J, et al. The tapetal AHL family protein TEK determines nexine formation in the pollen wall [J]. Nat Commun, 2014, 5: 3855.
[31]	Phan HA, Iacuone S, Li SF, et al. The MYB80 transcription factor is required for pollen development and the regulation of tapetal programmed cell death in Arabidopsis thaliana [J]. Plant Cell, 2011, 23(6): 2209-2224.
[32]	Zhu J, Lou Y, Xu XF, et al. A genetic pathway for tapetum development and function in Arabidopsis [J]. J Integr Plant Biol, 2011, 53(11): 892-900.
[33]	Ghelli R, Brunetti P, Marzi D, et al. The full-length auxin response factor 8 isoform ARF8.1 controls pollen cell wall formation and directly regulates TDF1, AMS and MS188 expression [J]. Plant J, 2023, 113(4): 851-865.
[34]	Ko SS, Li MJ, Ho YC, et al. Rice transcription factor GAMYB modulates bHLH142 and is homeostatically regulated by TDR during anther tapetal and pollen development [J]. J Exp Bot, 2021, 72(13): 4888-4903.
[35]	Lu JY, Xiong SX, Yin WZ, et al. MS1 a direct target of MS188 regulates the expression of key sporophytic pollen coat protein genes in Arabidopsis [J]. J Exp Bot, 2020, 71(16): 4877-4889.
[36]	Millar AA, Gubler F. The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development [J]. Plant Cell, 2005, 17(3): 705-721.
[37]	朱骏, 杨俊, 王晨, 等. 拟南芥转录因子AtMYB103的转录激活域鉴定 [J]. 上海交通大学学报: 农业科学版, 2009, 27(6): 611-614.
	Zhu J, Yang J, Wang C, et al. Characterization of activation domain of the Arabidopsis transcription factor AtMYB103 [J]. J Shanghai Jiao Tong Univ Agric Sci, 2009, 27(6): 611-614.