辣椒Whirly基因家族的鉴定及表达分析
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摘要
为了探索辣椒Whirly基因家族成员的功能和进化关系,通过生物信息学分析了其基因结构、保守基序、进化关系及表达模式,通过荧光定量PCR测定其在脱落酸、高温、低温、疫病等胁迫下的表达量。结果表明,从辣椒CM334中鉴定了2个Whirly基因家族成员,CaWHY1和CaWHY2。辣椒CaWHY1和CaWHY2同其他物种Whirly基因理化性质相比,差别不大,结构相似性高,CaWHY2基因和番茄的SlWHY2基因结构完全一样,在系统进化树中聚在一起。CaWHY1与SlWHY1聚在一起,但它们的基因结构不同,说明Whirly蛋白在进化过程中,结构保守。表达模式分析发现,CaWHY1和CaWHY2在辣椒CM334中均能表达,但在不同组织和果实发育时期的表达水平存在差异。CaWHY1和CaWHY2在不同胁迫处理下受到不同程度的诱导,其中CaWHY1明显受疫病的诱导,CaWHY2在低温处理和脱落酸胁迫下的表达量变化趋势相反。由此可知,Whirly基因家族在辣椒的生长发育中起调控作用,在各种逆境胁迫中也发挥一定作用。
To preliminarily explore the functions and evolutionary relations of Whirly gene family members, the bioinformatics was used to analyze the gene structure, conserved motif, evolutionary relationship and expression patterns, etc., and the fluorescence quantitative PCR was used to determine the expression levels under abscisic acid, high temperature, low temperature and epidemic stress. The results showed that two Whirly genes(CaWHY1 and CaWHY2) were identified from the pepper variety CM334. Compared with the physicochemical properties of other species, Whirly, the CaWHY1 and CaWHY2 have little difference in structure and similarity.The CaWHY2 gene had exactly the same genetic structure with the SlWHY2 gene of tomato, which were clustered together in phylogenetic tree. CaWHY1 and SlWHY1 were also clustered together, but their genetic structures were different, indicating that the structure of the Whirly protein is conserved during the evolution.The expression pattern analysis showed that both CaWHY1 and CaWHY2 could express in pepper CM334, but their expression levels were different in different tissues and at different fruit development stages. CaWHY1 and CaWHY2 were induced to different degrees under different stress treatments, among which CaWHY1 was obviously induced by epidemic diseases. The expression of CaWHY2 under low temperature and abscisic acid stress showed an opposite trend. Above results suggested that, Whirly gene family played a regulatory role in pepper growth and development, and also played a certaun role in various stress.
To preliminarily explore the functions and evolutionary relations of Whirly gene family members, the bioinformatics was used to analyze the gene structure, conserved motif, evolutionary relationship and expression patterns, etc., and the fluorescence quantitative PCR was used to determine the expression levels under abscisic acid, high temperature, low temperature and epidemic stress. The results showed that two Whirly genes(CaWHY1 and CaWHY2) were identified from the pepper variety CM334. Compared with the physicochemical properties of other species, Whirly, the CaWHY1 and CaWHY2 have little difference in structure and similarity.The CaWHY2 gene had exactly the same genetic structure with the SlWHY2 gene of tomato, which were clustered together in phylogenetic tree. CaWHY1 and SlWHY1 were also clustered together, but their genetic structures were different, indicating that the structure of the Whirly protein is conserved during the evolution.The expression pattern analysis showed that both CaWHY1 and CaWHY2 could express in pepper CM334, but their expression levels were different in different tissues and at different fruit development stages. CaWHY1 and CaWHY2 were induced to different degrees under different stress treatments, among which CaWHY1 was obviously induced by epidemic diseases. The expression of CaWHY2 under low temperature and abscisic acid stress showed an opposite trend. Above results suggested that, Whirly gene family played a regulatory role in pepper growth and development, and also played a certaun role in various stress.
引文
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[11] 闫秋艳,董飞,段增强,李汛,王嫒华,汤英.土壤温度对不同施肥方式下辣椒生长及土壤理化性质的影响[J].华北农学报,2018,33(2):195-201.doi:10.7668/hbnxb.2018.02.027.Yan Q Y,Dong F,Duan Z Q,Li X,Wang A H,Tang Y.Effect of soil temperature on soil physical-chemical properties and pepper growth in different fertilizer treatments[J].Acta Agriculturae Boreali-Sinica,2018,33(2):195-201.
[12] Xu Q,Dunbrack R L.Assignment of protein sequences to existing domain and family classification systems:Pfam and the PDB[J].Bioinformatics,2012,28(21):2763-2772.doi:10.1093/bioinformatics/bts533.
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[14] Barve A,Joshi B K.A smart source code editor for C[J].International Journal of Computer Science Engineering & Bioinformatics,2014,4(3):23-28.
[15] Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method[J].Methods,2001,25(4):402-408.doi:10.1006/meth.2001.1262.
[16] Bailey T L,Elkan C.Fitting a mixture model by expectation maximization to discover motifs in biopolymers[J].Proceedings International Conference on Intelligent Systems for Molecular Biology,1994,2:28-36.
[17] 孔凡英,邓永胜,周斌,孟庆伟.Whirly转录因子研究进展[J].植物生理学报,2012,48(7):643-653.doi:10.13592/j.cnki.ppj.2012.07.014.Kong F Y,Deng Y S,Zhou B,Meng Q W.Research advancement of Whirly transcription factors[J].Plant Physiology Journal,2012,48(7):643-653.
[18] 魏瑞敏,谢玲玲,欧阳娴,张亚利,戴雄泽,刘峰.辣椒ARF基因家族的鉴定与表达分析[J].西北植物学报,2017,37(6):1047-1058.doi:10.7606/j.issn.1000-4025.2017.06.1047.Wei R M,Xie L L,Ouyang X,Zhang Y L,Dai X Z,Liu F.Identification and expression analysis of ARF gene family in pepper[J].Acta Botanica Boreali-Occidentalia Sinica,2017,37(6):1047-1058.
[19] Grabowski E,Miao Y,Mulisch M,Krupinska K.Single stranded DNA-binding protein Whirly1 in barley leaves is located in plastids and the nucleus of the same cell[J].Plant Physiol,2008,147:1800-1804.doi:10.1104/pp.108.122796.
[20] Desveaux D,Marechal A,Brisson N.Whirly transcription factors:Defense gene regulation and beyond[J].Trends in Plant Science,2005,10(2):95-102.doi:10.1016/j.tplants.2004.12.008.
[21] Miller G,Suzuki N,Ciftci-yilmaz S,Mittler R.Reactive oxygen species homeostasis and signalling during drought and salinity stresses[J].Plant,Cell & Environment,2010,33(4):453-467.doi:10.1111/j.1365-3040.2009.02041.x.
[22] Chinnusamy V,Zhu J H,Zhu J K.Cold stress regulation of gene expression in plants[J].Trends in Plant Science,2007,12(10):444-451.doi:10.1016/j.tplants.2007.07.002.
[23] 杨洋,赖恭梯,赖钟雄.三明野生蕉Whirly转录因子的克隆及其在低温胁迫下的定量表达分析[J].热带作物学报,2014,35(8):1533-1538.doi:10.3969/j.issn.1000-2561.2014.08.015.Yang Y,Lai G T,Lai Z X.Cloning and expression under low temperature conditions of Whirly transcription factor in a wild banana accession (Musa spp.) from Sanming city[J].Chinese Journal of Tropical Crops,2014,35(8):1533-1538.
[2] Desveaux D,Despres C,Joyeux A,Subramaniam R,Brisson N.PBF-2 is a novel single-stranded DNA binding factor implicated in PR-10a gene activation in potato[J].The Plant Cell,2000,12(8):1477-1489.doi:10.1105/tpc.12.8.1477.
[3] Desveaux D,Marechal A,Brisson N.Whirly transcription factors:Defense gene regulation and beyond[J].Trends Plant Sci,2005,10(2):95-102.doi:10.1016/j.tplants.2004.12.008.
[4] Desveaux D,Subramaniam R,Despres C,Mess J N,Levesque C,Fobert P R.A "Whirly" transcription factor is required for salicylic acid-dependent disease resistance in Arabidopsis[J].Developmental Cell,2004,6(2):229-240.doi:10.1016/s1534-5807(04)00028-0.
[5] Yoo H H,Kwon C,Lee M M,Chung I K.Single-stranded DNA binding factor AtWHY1 modulates telomere length homeostasis in Arabidopsis [J].The Plant Journal,2007,49(3):442-451.doi:10.1111/j.1365-313X.2006.02974.x.
[6] Marechal A,Parent J S,Veronneau-lafortune F,Joyeux A,Lang B F,Brisson N.Whirly proteins maintain plastid genome stability in Arabidopsis[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106(34):14693-14698.doi:10.1073/pnas.0901710106.
[7] Cappadocia L,Marechal A,Parent J S,Lepage E,Sygusch J,Brisson N.Crystal structures of DNA-Whirly complexes and their role in Arabidopsis organelle genome repair[J].The Plant Cell,2010,22(6):1849-1867.doi:10.1105/tpc.109.071399.
[8] Kim S,Park M,Yeom S I,Kim Y M,Lee J M,Lee H A.Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species[J].Nature Genetics,2014,46(3):270-278.doi:10.1038/ng.2877.
[9] 魏小春,姚秋菊,原玉香,赵艳艳,王志勇,姜俊,蒋武生,张晓伟.辣椒CaWRKY13基因克隆及非生物胁迫下表达分析[J].分子植物育种,2016,14(10):2582-2588.doi:10.13271/j.mpb.014.002582.Wei X C Yao Q J,Yuan Y X,Zhao Y Y,Wang Z Y,Jiang J,Jiang W S,Zhang X W.Cloning and expression analysis of CaWRKY13 gene from Capsicum annuum L.under abiotic stress[J].Molecular Plant Breeding,2016,14(10):2582-2588.
[10] 魏小春,李艳,姚秋菊,原玉香,赵艳艳,王志勇,姜俊,段俊枝,蒋武生,张晓伟.辣椒CaCBF1A基因的克隆及非生物胁迫下表达分析[J].河南农业科学,2016,45(12):110-115.doi:10.15933/j.cnki.1004-3268.2016.12.022.Wei X C,Li Y,Yao Q J,Yuan Y X,Zhao Y Y,Wang Z Y,Jiang J,Duan J Z,Jiang W S,Zhang X W.Cloning and expression analysis of CaCBF1A gene from Capsicum annuum L.under abiotic stress[J].Journal of Henan Agricultural Sciences,2016,45(12):110-115.
[11] 闫秋艳,董飞,段增强,李汛,王嫒华,汤英.土壤温度对不同施肥方式下辣椒生长及土壤理化性质的影响[J].华北农学报,2018,33(2):195-201.doi:10.7668/hbnxb.2018.02.027.Yan Q Y,Dong F,Duan Z Q,Li X,Wang A H,Tang Y.Effect of soil temperature on soil physical-chemical properties and pepper growth in different fertilizer treatments[J].Acta Agriculturae Boreali-Sinica,2018,33(2):195-201.
[12] Xu Q,Dunbrack R L.Assignment of protein sequences to existing domain and family classification systems:Pfam and the PDB[J].Bioinformatics,2012,28(21):2763-2772.doi:10.1093/bioinformatics/bts533.
[13] Artimo P,Jonnalagedda M,Arnold K,Baratin D,Csardi G,de Castro E.ExPASy:SIB bioinformatics resource portal[J].Nucleic Acids Research,2012,40:597-603.doi:10.1093/nar/gks400.
[14] Barve A,Joshi B K.A smart source code editor for C[J].International Journal of Computer Science Engineering & Bioinformatics,2014,4(3):23-28.
[15] Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method[J].Methods,2001,25(4):402-408.doi:10.1006/meth.2001.1262.
[16] Bailey T L,Elkan C.Fitting a mixture model by expectation maximization to discover motifs in biopolymers[J].Proceedings International Conference on Intelligent Systems for Molecular Biology,1994,2:28-36.
[17] 孔凡英,邓永胜,周斌,孟庆伟.Whirly转录因子研究进展[J].植物生理学报,2012,48(7):643-653.doi:10.13592/j.cnki.ppj.2012.07.014.Kong F Y,Deng Y S,Zhou B,Meng Q W.Research advancement of Whirly transcription factors[J].Plant Physiology Journal,2012,48(7):643-653.
[18] 魏瑞敏,谢玲玲,欧阳娴,张亚利,戴雄泽,刘峰.辣椒ARF基因家族的鉴定与表达分析[J].西北植物学报,2017,37(6):1047-1058.doi:10.7606/j.issn.1000-4025.2017.06.1047.Wei R M,Xie L L,Ouyang X,Zhang Y L,Dai X Z,Liu F.Identification and expression analysis of ARF gene family in pepper[J].Acta Botanica Boreali-Occidentalia Sinica,2017,37(6):1047-1058.
[19] Grabowski E,Miao Y,Mulisch M,Krupinska K.Single stranded DNA-binding protein Whirly1 in barley leaves is located in plastids and the nucleus of the same cell[J].Plant Physiol,2008,147:1800-1804.doi:10.1104/pp.108.122796.
[20] Desveaux D,Marechal A,Brisson N.Whirly transcription factors:Defense gene regulation and beyond[J].Trends in Plant Science,2005,10(2):95-102.doi:10.1016/j.tplants.2004.12.008.
[21] Miller G,Suzuki N,Ciftci-yilmaz S,Mittler R.Reactive oxygen species homeostasis and signalling during drought and salinity stresses[J].Plant,Cell & Environment,2010,33(4):453-467.doi:10.1111/j.1365-3040.2009.02041.x.
[22] Chinnusamy V,Zhu J H,Zhu J K.Cold stress regulation of gene expression in plants[J].Trends in Plant Science,2007,12(10):444-451.doi:10.1016/j.tplants.2007.07.002.
[23] 杨洋,赖恭梯,赖钟雄.三明野生蕉Whirly转录因子的克隆及其在低温胁迫下的定量表达分析[J].热带作物学报,2014,35(8):1533-1538.doi:10.3969/j.issn.1000-2561.2014.08.015.Yang Y,Lai G T,Lai Z X.Cloning and expression under low temperature conditions of Whirly transcription factor in a wild banana accession (Musa spp.) from Sanming city[J].Chinese Journal of Tropical Crops,2014,35(8):1533-1538.