本氏烟BOI家族基因的全基因组鉴定与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0789

摘要/Abstract

摘要：

目的明确RING型泛素E3连接酶灰霉病易感互作因子（botrytis susceptible1 interactor, BOI）在赤霉素信号传导和黄瓜绿斑驳花叶病毒（cucumber green mottle mosaic virus, CGMMV）侵染过程中的作用及其基因家族特征，为理解茄科作物抗病毒分子机制及化学调控提供理论依据。方法通过生物信息学对本氏烟BOI基因家族进行系统进化关系、基因结构、保守基序、启动子顺式作用元件、转录因子及组织表达特征进行分析，利用荧光定量PCR验证NbBOI家族成员在生物胁迫和激素处理下的表达变化，并通过酵母双杂交试验筛选出与CGMMV编码蛋白互作的BOI家族基因。结果本氏烟BOI基因家族有13个成员，可划分为4个亚家族；同一进化分支的BOI家族成员的Motif及基因结构具有高度的一致性；启动子顺式作用元件预测筛选到6个与赤霉素通路相关基因；通过荧光定量PCR检测发现，NbBOI家族成员在组织特异性表达模式上存在差异，并且对病毒侵染和赤霉素处理的响应模式不同；酵母双杂交验证了NbBRG2.1与CGMMV编码的外壳蛋白CP、运动蛋白MP相互作用。结论本氏烟BOI基因家族具有高度的保守性，参与植物的生物胁迫防御和激素信号传导，其中NbBRG1.2在赤霉素合成途径中起重要作用，NbBOI.1、NbBOI.2、NbBOI.3、NbBRG1.2、NbBRG2.1、NbBRG2.3在植物对CGMMV侵染的防御机制中起一定作用。在病毒侵染过程中，通过病毒蛋白劫持寄主内NbBRG2.1，从而破坏寄主内激素平衡。

关键词: 本氏烟, 灰霉病易感互作因子, 组织特异性, 病毒侵染, 赤霉素信号转导

Abstract:

Objective To clarify the roles of RING-type ubiquitin E3 ligase Botrytis Susceptible1 Interactor (BOI) in gibberellin (GA) signal transduction and cucumber green mottle mosaic virus (CGMMV) infection, as well as the characteristics of its gene family, thus to provide a theoretical basis for understanding the molecular mechanisms of antiviral defense in Solanaceae crops and their chemical regulation. Method Bioinformatics approaches were used to analyze the phylogenetic relationships, gene structure, conserved motifs, promoter cis-acting elements, transcription factors, and tissue expression characteristics of NbBOI, and fluorescence quantitative PCR was applied to verify the expression changes of NbBOI family members under biotic stress and hormone treatments, and yeast two-hybrid assay was employed to screen out the BOI family genes that interacted with CGMMV encoded proteins. Result A total of thirteen BOI genes were identified in Nicotiana benthamiana, and they were divided into four subfamilies. The Motifs and gene structures of BOI family members in the same evolutionary branch were highly consistent. Six genes related to the gibberellin pathway were identified through prediction and promoter cis-acting elements analysis. Fluorescence quantitative PCR detection showed that members of the NbBOI family had differences in tissue-specific expression patterns and revealed different response patterns to virus infection and gibberellin treatment. The yeast two-hybrid assay verified that NbBRG2.1 interacted with the coat protein (CP) and movement protein encoded by CGMMV. Conclusion The BOI gene family in N. benthamiana shows high conservation and is involved in plant biotic stress defense and hormone signal transduction. Among its members, NbBRG1.2 plays a crucial role in the gibberellin (GA) biosynthesis pathway, while NbBOI.1, NbBOI.2, NbBOI.3, NbBRG1.2, NbBRG2.1, and NbBRG2.3 play a certain role in the defense mechanism of plants against CGMMV infection. During the viral infection process, viral proteins hijack the NbBRG2.1, thereby disrupting the hormonal balance in the host.

Key words: Nicotiana benthamiana, botrytis susceptible1 interactor, tissue specificity, virus infection, gibberellin signal transduction

付涵, 孙书豪, 张思晴, 艾妞, 于杨, 于连伟, 王琼琼, 韩晓玉, 施艳, 韩卫丽, 杨雪. 本氏烟BOI家族基因的全基因组鉴定与表达分析[J]. 生物技术通报, 2026, 42(2): 207-217.

FU Han, SUN Shu-hao, ZHANG Si-qing, AI Niu, YU Yang, YU Lian-wei, WANG Qiong-qiong, HAN Xiao-yu, SHI Yan, HAN Wei-li, YANG Xue. Genome-wide Identification and Expression Analysis of the BOI Gene Family in Nicotiana benthamiana[J]. Biotechnology Bulletin, 2026, 42(2): 207-217.

图/表 11

参考文献 31

[1]	Kawasaki T, Nam J, Boyes DC, et al. A duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and RPS2-mediated hypersensitive response [J]. Plant J, 2005, 44(2): 258-270.
[2]	Li ZW, Huang JZ, Hu Y, et al. The RING-type E3 ligase BOI interacts with EXO70E2 and mediates its ubiquitination in Arabidopsis [J]. Life, 2024, 14(9): 1169.
[3]	Luo HL, Laluk K, Lai ZB, et al. The Arabidopsis botrytis Susceptible1 interactor defines a subclass of RING E3 ligases that regulate pathogen and stress responses [J]. Plant Physiol, 2010, 154(4): 1766-1782.
[4]	Sun C, Yao GF, Li LX, et al. E3 ligase BRG3 persulfidation delays tomato ripening by reducing ubiquitination of the repressor WRKY71 [J]. Plant Physiol, 2023, 192(1): 616-632.
[5]	罗红丽, 宋凤鸣, Mengiste T. 拟南芥BOI基因在抗氧化胁迫和细胞死亡中的功能分析 [J]. 植物病理学报, 2012, 42(1): 65-72.
	Luo HL, Song FM, Mengiste T. Functional analysis of Arabidopsis botrytis Susceptible 1 Interactor in oxidative stress and programmed cell death [J]. Acta Phytopathol Sin, 2012, 42(1): 65-72.
[6]	张萍. BnaBOI1与BnaPATL2互作调控油菜分枝数的分子机理研究 [D]. 重庆: 西南大学, 2022.
	Zhang P. Molecular mechanism for regulating branch number in Brassica napus via interactions between BnaBOI1 and BnaPATL2 [D]. Chongqing: Southwest University, 2022.
[7]	Huang JZ, Wu XQ, Gao ZY. The RING-type protein BOI negatively regulates the protein level of a CC-NBS-LRR in Arabidopsis [J]. Biochem Biophys Res Commun, 2021, 578: 104-109.
[8]	Nguyen KT, Park J, Park E, et al. The Arabidopsis RING domain protein BOI inhibits flowering via CO-dependent and CO-independent mechanisms [J]. Mol Plant, 2015, 8(12): 1725-1736.
[9]	Park J, Nguyen KT, Park E, et al. DELLA proteins and their interacting RING finger proteins repress gibberellin responses by binding to the promoters of a subset of gibberellin-responsive genes in Arabidopsis [J]. Plant Cell, 2013, 25(3): 927-943.
[10]	陈智华, 乔振升, 李嘉其, 等. 滇杨TCP基因家族的全基因组鉴定与分析 [J]. 生物技术通报, 2024, 40(11): 214-226.
	Chen ZH, Qiao ZS, Li JQ, et al. Genome-wide identification and analysis of the TCP gene family in Populus yunnanensis [J]. Biotechnol Bull, 2024, 40(11): 214-226.
[11]	吴浩, 董伟峰, 贺子天, 等. 水稻BXL基因家族的全基因组鉴定及表达分析 [J]. 生物技术通报, 2025(6): 87-98.
	Wu H, Dong WF, He ZT, et al. Genome-wide identification and expression analysis of the Rice BXL Gene Family [J]. Biotechnology Bulletin, 2025(6): 87-98.
[12]	Letunic I, Bork P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool [J]. Nucleic Acids Res, 2024, 52(W1): W78-W82.
[13]	Ma WP, Liu X, Chen K, et al. Genome-wide re-identification and analysis of CrRLK1Ls in tomato [J]. Int J Mol Sci, 2023, 24(4): 3142.
[14]	吴娅, 姚润, 杨含婷, 等. 凤梨薄荷SDR基因家族全基因组鉴定及表达分析 [J]. 生物技术通报, 2025, 41(5): 175-185.
	Wu Y, Yao R, Yang HT, et al. Genome-wide identification and expression analysis of SDR gene family in Mentha suaveolens ‘variegata’ [J]. Biotechnol Bull, 2025, 41(5): 175-185.
[15]	Rao SF, Wu XY, Zheng HY, et al. Genome-wide identification and analysis of Catharanthus roseus RLK1-like kinases in Nicotiana benthamiana [J]. BMC Plant Biol, 2021, 21(1): 425.
[16]	于连伟, 姜兴林, 杨灵玲, 等. 转录因子NbERF RAP2-1在黄瓜绿斑驳花叶病毒侵染中的功能 [J]. 中国农业科学, 2023, 56(15): 2919-2928.
	Yu LW, Jiang XL, Yang LL, et al. Function of transcription factor NbERF RAP2-1 in cucumber green mottle mosaic virus infection [J]. Sci Agric Sin, 2023, 56(15): 2919-2928.
[17]	Yang X, Jiang XL, Fu H, et al. Cucumber green mottle mosaic virus coat protein hijacks mitochondrial ATPδ to promote viral infection [J]. Mol Plant Pathol, 2024, 25(11): e70034.
[18]	Huang JZ, Wu XQ, Gao ZY. A nucleocytoplasmic-localized E3 ligase affects the NLR receptor stability [J]. Biochem Biophys Res Commun, 2021, 583: 1-6.
[19]	Wang SS, Wang YJ. Harnessing hormone gibberellin knowledge for plant height regulation [J]. Plant Cell Rep, 2022, 41(10): 1945-1953.
[20]	Guo SQ, Chen YX, Ju YL, et al. Fine-tuning gibberellin improves rice alkali-thermal tolerance and yield [J]. Nature, 2025, 639(8053): 162-171.
[21]	戴琪. 赤霉素结合2, 4-表油菜素内酯处理对缓解冷藏桃果实冷害的机制研究 [D]. 沈阳: 沈阳农业大学, 2023.
	Dai Q. Research on the mechanism of gibberellin combined with 2,4 epibrassinolide treatment on alleviating cold damage of chilling peach fruits [D]. Shenyang: Shenyang Agricultural University, 2023.
[22]	Huang JS, Xie B, Xian FJ, et al. Gibberellin signalling mediates nucleocytoplasmic trafficking of Sucrose Synthase 1 to regulate the drought tolerance in rice [J]. Plant Biotechnol J, 2025, 23(6): 1909-1926.
[23]	Bao SJ, Hua CM, Shen LS, et al. New insights into gibberellin signaling in regulating flowering in Arabidopsis [J]. J Integr Plant Biol, 2020, 62(1): 118-131.
[24]	Yimer HZ, Nahar K, Kyndt T, et al. Gibberellin antagonizes jasmonate-induced defense against Meloidogyne graminicola in rice [J]. New Phytol, 2018, 218(2): 646-660.
[25]	Li PB, Guo LM, Lang XY, et al. Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation, to promote viral infection and symptom development in N. benthamiana [J]. PLoS Pathog, 2022, 18(4): e1010217.
[26]	Navarro L, Bari R, Achard P, et al. DELLAs control plant immune responses by modulating the balance of jasmonic acid and salicylic acid signaling [J]. Curr Biol, 2008, 18(9): 650-655.
[27]	孔明悦. 柑橘木虱胁迫及喷施赤霉素和微肥对柑橘生理生化指标的影响 [D]. 佛山: 佛山科学技术学院, 2023.
	Kong MY. Effects of Diaphorina citri,gibberellin and microfertilizer on hysiological and biochemical indices of citrus [D]. Foshan: Foshan University, 2023.
[28]	Smalle J, Vierstra RD. The ubiquitin 26S proteasome proteolytic pathway [J]. Annu Rev Plant Biol, 2004, 55: 555-590.
[29]	Dong CH, Agarwal M, Zhang YY, et al. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1 [J]. Proc Natl Acad Sci USA, 2006, 103(21): 8281-8286.
[30]	Ling KS, Li R, Zhang W. First report of Cucumber green mottle mosaic virus infecting greenhouse cucumber in Canada [J]. Plant Dis, 2014, 98(5): 701.
[31]	姜兴林, 于连伟, 付涵, 等. 转录因子NbMYB1R1通过促进活性氧积累抑制病毒侵染 [J]. 中国农业科学, 2024, 57(8): 1490-1505.
	Jiang XL, Yu LW, Fu H, et al. The transcription factor NbMYB1R1 inhibits viral infection by promoting ROS accumulation [J]. Sci Agric Sin, 2024, 57(8): 1490-1505.

基因名称 Gene name	上游引物 Forward primer （5'-3'）	下游引物 Reverse primer （5'-3'）
NbBOI.1	CGGGTGTAACGAGAGAGACG	AACACACAAATGGCGACACG
NbBOI.2	CTAGTCAATCATCGGCTTTCCA	TAGGCGTGCCATTGTTTCCT
NbBOI.3	TTGACTTAACCCAGCTCCAC	AGAATGATGTTGTTTTTGTTGGTGC
NbBRG1.2	CAACAGCAGTTGCAAAAAGACAA	GCGTCAATCTCAATCGCTCC
NbBRG2.1	TAGCTCCAGGTCCAGCTGAT	ACCCAAACGTCCATTATCCA
NbBRG2.3	ACTATTACACCACCGCCACTT	GGCATCCAAATTGCAGCTGAG

基因名称 Gene name	上游引物 Forward primer （5'-3'）	下游引物 Reverse primer （5'-3'）
NbBOI.1	CGGGTGTAACGAGAGAGACG	AACACACAAATGGCGACACG
NbBOI.2	CTAGTCAATCATCGGCTTTCCA	TAGGCGTGCCATTGTTTCCT
NbBOI.3	TTGACTTAACCCAGCTCCAC	AGAATGATGTTGTTTTTGTTGGTGC
NbBRG1.2	CAACAGCAGTTGCAAAAAGACAA	GCGTCAATCTCAATCGCTCC
NbBRG2.1	TAGCTCCAGGTCCAGCTGAT	ACCCAAACGTCCATTATCCA
NbBRG2.3	ACTATTACACCACCGCCACTT	GGCATCCAAATTGCAGCTGAG

基因名 Gene name	基因ID Gene ID	氨基酸数 Protein length （aa）	分子量 Molecular weight （kD）	等电点 pI	不稳定指数Instability index	总平均亲水系数Grand average of hydropathicity	脂肪指数 Aliphatic index	亚细胞定位 Subcellular location
NbBRG2.1	Niben101Scf04995g03009.1	337	38 311.27	5.23	48.590	-0.597	83.89	Nucleus
NbBRG2.2	Niben101Scf05551g00006.1	579	65 546.86	6.53	43.220	-0.643	75.82	Nucleus
NbBRG2.3	Niben101Scf09599g00015.1	361	40 133.59	7.49	36.470	-0.487	81.52	Nucleus
NbBRG2.4	Niben101Scf00468g01013.1	347	38 916.32	7.88	39.380	-0.493	74.21	Nucleus
NbBRG3.1	Niben101Scf25562g00007.1	327	35 967.52	5.71	49.640	-0.343	84.16	Nucleus
NbBRG3.2	Niben101Scf02156g11001.1	349	38 832.05	5.89	43.620	-0.464	77.91	Nucleus
NbBRG3.3	Niben101Scf04499g00008.1	350	39 076.28	5.9	47.580	-0.476	78.26	Nucleus
NbBRG3.4	Niben101Scf01455g00005.1	431	47 614.25	5.09	41.520	-0.472	77.42	Nucleus
NbBRG1.1	Niben101Scf06106g01018.1	283	32 891.4	5.92	63.690	-0.632	77.53	Nucleus
NbBRG1.2	Niben101Scf07614g02001.1	269	31 231.47	7.92	72.720	-0.715	65.32	Nucleus
NbBOI.1	Niben101Scf04159g01016.1	316	34 681.7	6.52	68.610	-0.671	66.2	Nucleus
NbBOI.2	Niben101Scf01526g12008.1	338	37 295.86	6.24	64.790	-0.501	79.44	Nucleus
NbBOI.3	Niben101Scf06847g02001.1	339	37 640.29	7.2	67.220	-0.561	79.23	Nucleus

基因名 Gene name	基因ID Gene ID	氨基酸数 Protein length （aa）	分子量 Molecular weight （kD）	等电点 pI	不稳定指数Instability index	总平均亲水系数Grand average of hydropathicity	脂肪指数 Aliphatic index	亚细胞定位 Subcellular location
NbBRG2.1	Niben101Scf04995g03009.1	337	38 311.27	5.23	48.590	-0.597	83.89	Nucleus
NbBRG2.2	Niben101Scf05551g00006.1	579	65 546.86	6.53	43.220	-0.643	75.82	Nucleus
NbBRG2.3	Niben101Scf09599g00015.1	361	40 133.59	7.49	36.470	-0.487	81.52	Nucleus
NbBRG2.4	Niben101Scf00468g01013.1	347	38 916.32	7.88	39.380	-0.493	74.21	Nucleus
NbBRG3.1	Niben101Scf25562g00007.1	327	35 967.52	5.71	49.640	-0.343	84.16	Nucleus
NbBRG3.2	Niben101Scf02156g11001.1	349	38 832.05	5.89	43.620	-0.464	77.91	Nucleus
NbBRG3.3	Niben101Scf04499g00008.1	350	39 076.28	5.9	47.580	-0.476	78.26	Nucleus
NbBRG3.4	Niben101Scf01455g00005.1	431	47 614.25	5.09	41.520	-0.472	77.42	Nucleus
NbBRG1.1	Niben101Scf06106g01018.1	283	32 891.4	5.92	63.690	-0.632	77.53	Nucleus
NbBRG1.2	Niben101Scf07614g02001.1	269	31 231.47	7.92	72.720	-0.715	65.32	Nucleus
NbBOI.1	Niben101Scf04159g01016.1	316	34 681.7	6.52	68.610	-0.671	66.2	Nucleus
NbBOI.2	Niben101Scf01526g12008.1	338	37 295.86	6.24	64.790	-0.501	79.44	Nucleus
NbBOI.3	Niben101Scf06847g02001.1	339	37 640.29	7.2	67.220	-0.561	79.23	Nucleus