甘蓝型油菜Metacaspase家族基因鉴定及其对干旱胁迫响应分析

doi:10.7668/hbnxb.20195788

摘要/Abstract

摘要：

Metacaspase(MC)是一种精氨酸/苏氨酸特异性蛋白酶,研究表明其在细胞程序性死亡中发挥作用。为探究 MC家族基因在甘蓝型油菜基因组中的分布及其是否响应干旱胁迫,对甘蓝型油菜MC家族基因成员的理化性质、系统发育、基因结构、保守结构域、顺式作用元件及干旱(PEG6000)和脱落酸(ABA)胁迫下的表达模式进行系统分析。共鉴定到25个BnMC基因,染色体定位显示,25个BnMC分布于13条染色体上,17个成员定位于细胞核,7个成员定位在细胞质;系统进化树将BnMC分为2个大类(Ⅰ型和Ⅱ型),4个分支(Group A、Group B、Group C和Group D),同一分支的BnMC具有相似的基因结构和保守基序分布。MC家族基因的核心启动子区包含的顺式作用元件有光响应元件、植物激素响应元件、植物生长发育响应元件和胁迫响应元件,非生物胁迫响应相关的所有顺式作用元件中,脱落酸响应元件(ABRE)数目最多,为79个,所有成员都含有该元件。转录组学结果分析发现,干旱胁迫后BnMC10、BnMC22、BnMC1、BnMC12、BnMC8的表达量上调,BnMC4和BnMC5表达下调。qRT-PCR检测显示,BnMC10、BnMC8、BnMC1、BnMC12在根和叶中均有表达,且受PEG6000和ABA诱导上调,其中BnMC1受诱导上调最为显著。综上所述,甘蓝型油菜响应干旱胁迫涉及对MC家族基因的表达水平调控。

关键词: 甘蓝型油菜, Metacaspase, 基因家族, 干旱胁迫

Abstract:

Metacaspase (MC) belongs to arginine/threonine specific protease,studies have shown that it plays a role in programmed cell death.To investigate the distribution of MC family genes in the genome of Brassica napus and whether they respond to drought stress,this study systematically analyzed the physicochemical properties,phylogeny,gene structure,conserved domains,cis-acting elements,and expression patterns of MC family genes under drought(PEG6000)and abscisic acid(ABA)stress in B.napus.A total of 25 BnMC genes were identified.Chromosomal localization showed that the 25 BnMCs were distributed on 13 chromosomes.Subcellular localization prediction showed that 17 members of the BnMC family were localized in the nucleus and seven members were in the cytoplasm.The phylogenetic tree classified BnMC into two major classes (Type Ⅰ and Type Ⅱ) and four branches (Group A,Group B,Group C,and Group D).BnMCs of the same branch had similar gene structure and conserved motif distribution.The core promoter regions of BnMC contained four types of cis-acting elements:light response element,phytohormone response element,plant growth and development response element and stress response element.Among all the cis-acting elements related to abiotic stress responses,the abscisic acid response element (ABRE) was the most abundant,with a total of 79.All members contained this cis-acting element.The transcriptome sequencing revealed that the expressions of BnMC10,BnMC22,BnMC1,BnMC12 and BnMC8 were up-regulated and the expressions of BnMC4 and BnMC5 were down-regulated after drought treatment.The qRT-PCR assay showed BnMC10,BnMC8,BnMC1 and BnMC12 genes were expressed in both roots and leaves and were up-regulated by both PEG6000 and ABA,with BnMC1 showing the most significant up-regulating changes.In summary,the response of B.napus to drought stress involves the regulation of the expression level of MC family genes.

Key words: Brassica napus, Metacaspase, Gene family, Drought stress

中图分类号:

李天, 王道杰, 张晓娟, 侯扬子. 甘蓝型油菜Metacaspase家族基因鉴定及其对干旱胁迫响应分析[J]. 华北农学报, 2025, 40(6): 21-30. doi: 10.7668/hbnxb.20195788.

LI Tian, WANG Daojie, ZHANG Xiaojuan, HOU Yangzi. Identification of Metacaspase Family Genes in Brassica napus and Analysis of Gene Expression under Drought Stress[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(6): 21-30. doi: 10.7668/hbnxb.20195788.

http://www.hbnxb.net/CN/Y2025/V40/I6/21

图/表 8

参考文献 33

[1]	Huh S U. Evolutionary diversity and function of metacaspases in plants:similar to but not caspases[J]. International Journal of Molecular Sciences, 2022, 23(9):4588.doi:10.3390/ijms23094588. URL
[2]	Tsiatsiani L, Van Breusegem F, Gallois P, Zavialov A, Lam E, Bozhkov P V. Metacaspases[J]. Cell Death & Differentiation, 2011, 18(8):1279-1288.doi:10.1038/cdd.2011.66.
[3]	Wang L K, Zhang H. Genomewide survey and characterization of metacaspase gene family in rice (Oryza sativa)[J]. Journal of Genetics, 2014, 93(1):93-102.doi:10.1007/s12041-014-0343-6. pmid: 24840826
[4]	Liu H, Liu J, Wei Y X. Identification and analysis of the metacaspase gene family in tomato[J]. Biochemical and Biophysical Research Communications, 2016, 479(3):523-529.doi:10.1016/j.bbrc.2016.09.103. pmid: 27664707
[5]	Fan S M, Liu A Y, Zhang Z, Zou X Y, Jiang X, Huang J Y, Fan L Q, Zhang Z B, Deng X Y, Ge Q, Gong W K, Li J W, Gong J W, Shi Y Z, Lei K, Zhang S Y, Jia T T, Zhang L P, Yuan Y L, Shang H H. Genome-wide identification and expression analysis of the metacaspase gene family in Gossypium species[J]. Genes, 2019, 10(7):527.doi:10.3390/genes10070527. URL
[6]	Coll N S, Epple P, Dangl J L. Programmed cell death in the plant immune system[J]. Cell Death & Differentiation, 2011, 18(8):1247-1256.doi:10.1038/cdd.2011.37.
[7]	Hoeberichts F A,ten Have A,Woltering E J. A tomato metacaspase gene is upregulated during programmed cell death in Botrytis cinerea-infected leaves[J]. Planta, 2003, 217(3):517-522.doi:10.1007/s00425-003-1049-9. pmid: 12783227
[8]	Zhou Y, Hu L F, Jiang L W, Liu S Q. Genome-wide identification,characterization,and transcriptional analysis of the metacaspase gene family in cucumber (Cucumis sativus)[J]. Genome, 2018, 61(3):187-194.doi:10.1139/gen-2017-0174. URL
[9]	Pitsili E, Rodriguez-Trevino R, Ruiz-Solani N, Demir F, Kastanaki E, Dambire C, de Pedro-Jové R, Vercammen D, Salguero-Linares J, Hall H, Mantz M, Schuler M, Tuominen H, Van Breusegem F, Valls M, Munn-Bosch S, Holdsworth M J, Huesgen P F, Rodriguez-Villalon A, Coll N S. A phloem-localized Arabidopsis metacaspase (AtMC3) improves drought tolerance[J]. New Phytologist, 2023, 239(4):1281-1299.doi:10.1111/nph.19022. URL
[10]	陈雨蝶, 张泽荣, 李恒湘, 李天乐, 曾思杰, 邬贤梦, 熊兴华, 肖钢. 甘蓝型油菜BnEXO基因家族功能与表达分析[J]. 华北农学报, 2024, 39(5):24-32.doi:10.7668/hbnxb.20194909.
	Chen Y D, Zhang Z R, Li H X, Li T L, Zeng S J, Wu X M, Xiong X H, Xiao G. Gene family function and expression analysis of BnEXO in Brassica napus[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5):24-32.
[11]	文雁成, 王汉中, 沈金雄, 刘贵华, 张书芬. 用SRAP标记分析中国甘蓝型油菜品种的遗传多样性和遗传基础[J]. 中国农业科学, 2006, 39(2):246-256.doi:10.3321/j.issn:0578-1752.2006.02.005.
	Wen Y C, Wang H Z, Shen J X, Liu G H, Zhang S F. Analysis of genetic diversity and genetic basis of Chinese rapeseed cultivars (Brassica napus L.) by sequence-related amplified polymorphism markers[J]. Scientia Agricultura Sinica, 2006, 39(2):246-256.
[12]	李积铭, 张耀文, 郭安强, 翟兰菊, 李和平, 李爱国. 油菜抗寒性和寒旱区油菜育种探析[J]. 华北农学报, 2023, 38(S1):131-144.doi:10.7668/hbnxb.20194305.
	Li J M, Zhang Y W, Guo A Q, Zhai L J, Li H P, Li A G. Advances in research on cold resistance of rapeseed and analysis of rapeseed breeding in cold and dry regions of China[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(S1):131-144. doi: 10.7668/hbnxb.20194305
[13]	Yang Z Q, Wang S B, Wei L L, Huang Y M, Liu D X, Jia Y P, Luo C F, Lin Y C, Liang C Y, Hu Y, Dai C, Guo L, Zhou Y M, Yang Q Y. BnIR:a multi-omics database with various tools for Brassica napus research and breeding[J]. Molecular Plant, 2023, 16(4):775-789.doi:10.1016/j.molp.2023.03.007. URL
[14]	Lamesch P, Berardini T Z, Li D H, Swarbreck D, Wilks C, Sasidharan R, Muller R, Dreher K, Alexander D L, Garcia-Hernandez M, Karthikeyan A S, Lee C H, Nelson W D, Ploetz L, Singh S, Wensel A, Huala E. The Arabidopsis information resource (TAIR):improved gene annotation and new tools[J]. Nucleic Acids Research,2012,40:D1202-D1210.doi:10.1093/nar/gkr1090.
[15]	Mistry J, Chuguransky S, Williams L, Qureshi M, Salazar G A, Sonnhammer E L L, Tosatto S C E, Paladin L, Raj S, Richardson L J, Finn R D, Bateman A. Pfam:the protein families database in 2021[J]. Nucleic Acids Research, 2021, 49(D1):D412-D419.doi:10.1093/nar/gkaa913.
[16]	Duvaud S, Gabella C, Lisacek F, Stockinger H, Ioannidis V, Durinx C. Expasy,the Swiss bioinformatics resource portal,as designed by its users[J]. Nucleic Acids Research, 2021, 49(W1):W216-W227.doi:10.1093/nar/gkab225.
[17]	Yu C S, Lin C J, Hwang J K. Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machines based on n-peptide compositions[J]. Protein Science, 2004, 13(5):1402-1406.doi:10.1110/ps.03479604. URL
[18]	Chen C J, Wu Y, Li J W, Wang X, Zeng Z H, Xu J, Liu Y L, Feng J T, Chen H, He Y H, Xia R. TBtools-II:a'one for all,all for one' bioinformatics platform for biological big-data mining[J]. Molecular Plant, 2023, 16(11):1733-1742.doi:10.1016/j.molp.2023.09.010. URL
[19]	Bailey T L, Johnson J, Grant C E, Noble W S. The MEME suite[J]. Nucleic Acids Research, 2015, 43(W1):W39-W49.doi:10.1093/nar/gkv416.
[20]	Rombauts S, Dehais P, Van Montagu M, Rouze P. PlantCARE,a plant cis-acting regulatory element database[J]. Nucleic Acids Research, 1999, 27(1):295-296.doi:10.1093/nar/27.1.295. pmid: 9847207
[21]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4):402-408.doi:10.1006/meth.2001.1262. pmid: 11846609
[22]	Shabalina S A, Ogurtsov A Y, Spiridonov A N, Novichkov P S, Spiridonov N A, Koonin E V. Distinct patterns of expression and evolution of intronless and intron-containing mammalian genes[J]. Molecular Biology and Evolution, 2010, 27(8):1745-1749.doi:10.1093/molbev/msq086. pmid: 20360214
[23]	Jain M, Khurana P, Tyagi A K, Khurana J P. Genome-wide analysis of intronless genes in rice and Arabidopsis[J]. Functional & Integrative Genomics, 2008, 8(1):69-78.doi:10.1007/s10142-007-0052-9.
[24]	Liu H, Lyu H M, Zhu K K, Van de Peer Y,Max Cheng Z M. The emergence and evolution of intron-poor and intronless genes in intron-rich plant gene families[J]. The Plant Journal, 2021, 105(4):1072-1082.doi:10.1111/tpj.15088. pmid: 33217085
[25]	Woltering E J. Death proteases:alive and kicking[J]. Trends in Plant Science, 2010, 15(4):185-188.doi:10.1016/j.tplants.2010.02.001. pmid: 20202889
[26]	Guo J Y, Wu Y L, Huang J Q, Yu K H, Chen M L, Han Y J, Zhong Z H, Lu G D, Hong Y H, Wang Z H, Chen X F. The Magnaporthe oryzae effector Avr-PikD suppresses rice immunity by inhibiting an LSD1-like transcriptional activator[J]. The Crop Journal, 2024, 12(2):482-492.doi:10.1016/j.cj.2024.01.011. URL
[27]	Sun S J, Ma W, Jia Z C, Ou C M, Li M L, Mao P S. Genomic identification and expression profiling of lesion simulating disease genes in alfalfa (Medicago sativa) elucidate their responsiveness to seed vigor[J]. Antioxidants, 2023, 12(9):1768.doi:10.3390/antiox12091768. URL
[28]	吴耀荣, 谢旗. ABA与植物胁迫抗性[J]. 植物学通报, 2006, 41(5):511-518.doi:10.3969/j.issn.1674-3466.2006.05.007.
	Wu Y R, Xie Q. ABA and plant stress response[J]. Chinese Bulletin of Botany, 2006, 41(5):511-518.
[29]	岑英, 余林婵, 劳子珊, 谭勇, 黄荣韶, 吴思滢, 姚绍嫦. 牛大力metacaspase基因CsMC1的克隆及超表达载体构建[J]. 分子植物育种, 2023, 21(13):4301-4310.doi:10.13271/j.mpb.021.004301.
	Cen Y, Yu L C, Lao Z S, Tan Y, Huang R S, Wu S Y, Yao S C. Cloning and over-expression vector constructed of CsMC1 gene from Callerya speciosa(champ.ex Benth.) schot[J]. Molecular Plant Breeding, 2023, 21(13):4301-4310.
[30]	Klemencˇicˇ M, Funk C. Evolution and structural diversity of metacaspases[J]. Journal of Experimental Botany, 2019, 70(7):2039-2047.doi:10.1093/jxb/erz082. pmid: 30921456
[31]	Chapin L J, Jones M L. A type I and a type II metacaspase are differentially regulated during Corolla development and in response to abiotic and biotic stresses in Petunia×hybrida[J]. Horticulturae, 2022, 8(12):1151.doi:10.3390/horticulturae8121151. URL
[32]	Yue J Y, Wang Y J, Jiao J L, Wang W W, Wang H Z. The metacaspase TaMCA-id negatively regulates salt-induced programmed cell death and functionally links with autophagy in wheat[J]. Frontiers in Plant Science, 2022,13:904933.doi:10.3389/fpls.2022.904933.
[33]	Zhu J K. Abiotic stress signaling and responses in plants[J]. Cell, 2016, 167(2):313-324.doi:10.1016/j.cell.2016.08.029. URL

基因名称 Gene name	正向引物序列(5'—3') Forward primer sequence(5'—3')	反向引物序列(5'—3') Reverse primer sequence(5'—3')
BnMC1	CAATCATTTTCCGCCGGCAC	GCAAGACTCGGGGAACTTG
BnMC8	GCACTTCCGGATAATGATGATGAAT	GCTTCATCTATGAGACCACCAC
BnMC10	TACCGTATCCCTAACAAGCAAAAC	GAAGTCCAGAGGACAGAGCGTC
BnMC12	ACGTGCCTTCGTCTTCTTTC	TGATGCACCCTTTCAACTCGT
BnActin	GCTGACCGTATGAGCAAAGA	CACGAACCAGAAGGCAGAAA

基因名称 Gene name	正向引物序列(5'—3') Forward primer sequence(5'—3')	反向引物序列(5'—3') Reverse primer sequence(5'—3')
BnMC1	CAATCATTTTCCGCCGGCAC	GCAAGACTCGGGGAACTTG
BnMC8	GCACTTCCGGATAATGATGATGAAT	GCTTCATCTATGAGACCACCAC
BnMC10	TACCGTATCCCTAACAAGCAAAAC	GAAGTCCAGAGGACAGAGCGTC
BnMC12	ACGTGCCTTCGTCTTCTTTC	TGATGCACCCTTTCAACTCGT
BnActin	GCTGACCGTATGAGCAAAGA	CACGAACCAGAAGGCAGAAA

基因名称 Gene name	参考基因ID Reference gene ID	氨基酸数目/aa Number of amino acids	分子质量/ku Molecular weight	等电点 Isoelectric point	亚细胞定位预测 Prediction of subcellular localization
BnMC1	BnaA01G0146400ZS	447	48.46	6.01	细胞核
BnMC2	BnaA02G0015400ZS	323	35.26	5.88	细胞质
BnMC3	BnaA02G0236900ZS	318	35.30	7.61	细胞核
BnMC4	BnaA02G0407600ZS	380	42.20	7.20	细胞核
BnMC5	BnaA06G0292800ZS	407	45.50	8.84	细胞核
BnMC6	BnaA07G0231500ZS	370	41.37	4.96	细胞质
BnMC7	BnaA07G0381800ZS	277	30.58	8.25	细胞核
BnMC8	BnaA07G0381900ZS	680	74.82	4.35	细胞核
BnMC9	BnaA09G0622400ZS	681	76.28	8.69	细胞核
BnMC10	BnaA09G0677200ZS	604	66.85	8.54	细胞外
BnMC11	BnaA10G0285700ZS	324	35.45	5.62	细胞质
BnMC12	BnaC01G0186200ZS	462	50.08	6.04	细胞核
BnMC13	BnaC02G0015500ZS	332	36.01	5.43	细胞质
BnMC14	BnaC02G0063600ZS	211	23.61	5.07	细胞核
BnMC15	BnaC02G0318400ZS	400	44.33	6.52	细胞核
BnMC16	BnaC02G0541600ZS	389	43.33	7.39	细胞核
BnMC17	BnaC03G0533600ZS	412	45.89	8.72	细胞核
BnMC18	BnaC05G0217400ZS	362	40.26	6.93	细胞核
BnMC19	BnaC05G0217600ZS	120	13.27	7.25	细胞质
BnMC20	BnaC06G0250200ZS	260	29.16	5.11	细胞质
BnMC21	BnaC06G0448900ZS	389	42.99	5.23	细胞核
BnMC22	BnaC06G0449100ZS	706	78.00	4.53	细胞核
BnMC23	BnaC08G0479000ZS	312	34.36	6.18	细胞核
BnMC24	BnaC09G0137300ZS	323	35.47	4.73	细胞核
BnMC25	BnaC09G0604300ZS	322	35.28	5.61	细胞质

基因名称 Gene name	参考基因ID Reference gene ID	氨基酸数目/aa Number of amino acids	分子质量/ku Molecular weight	等电点 Isoelectric point	亚细胞定位预测 Prediction of subcellular localization
BnMC1	BnaA01G0146400ZS	447	48.46	6.01	细胞核
BnMC2	BnaA02G0015400ZS	323	35.26	5.88	细胞质
BnMC3	BnaA02G0236900ZS	318	35.30	7.61	细胞核
BnMC4	BnaA02G0407600ZS	380	42.20	7.20	细胞核
BnMC5	BnaA06G0292800ZS	407	45.50	8.84	细胞核
BnMC6	BnaA07G0231500ZS	370	41.37	4.96	细胞质
BnMC7	BnaA07G0381800ZS	277	30.58	8.25	细胞核
BnMC8	BnaA07G0381900ZS	680	74.82	4.35	细胞核
BnMC9	BnaA09G0622400ZS	681	76.28	8.69	细胞核
BnMC10	BnaA09G0677200ZS	604	66.85	8.54	细胞外
BnMC11	BnaA10G0285700ZS	324	35.45	5.62	细胞质
BnMC12	BnaC01G0186200ZS	462	50.08	6.04	细胞核
BnMC13	BnaC02G0015500ZS	332	36.01	5.43	细胞质
BnMC14	BnaC02G0063600ZS	211	23.61	5.07	细胞核
BnMC15	BnaC02G0318400ZS	400	44.33	6.52	细胞核
BnMC16	BnaC02G0541600ZS	389	43.33	7.39	细胞核
BnMC17	BnaC03G0533600ZS	412	45.89	8.72	细胞核
BnMC18	BnaC05G0217400ZS	362	40.26	6.93	细胞核
BnMC19	BnaC05G0217600ZS	120	13.27	7.25	细胞质
BnMC20	BnaC06G0250200ZS	260	29.16	5.11	细胞质
BnMC21	BnaC06G0448900ZS	389	42.99	5.23	细胞核
BnMC22	BnaC06G0449100ZS	706	78.00	4.53	细胞核
BnMC23	BnaC08G0479000ZS	312	34.36	6.18	细胞核
BnMC24	BnaC09G0137300ZS	323	35.47	4.73	细胞核
BnMC25	BnaC09G0604300ZS	322	35.28	5.61	细胞质

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