水曲柳FmWRKY44基因克隆及表达分析
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摘要
克隆水曲柳FmWRKY44基因,探究其在非生物胁迫和激素胁迫中的作用。利用水曲柳干旱转录组序列设计特异引物,克隆FmWRKY44基因的完整ORF序列,并对该序列及其编码产物进行生物信息学分析,采用qRTPCR技术分析FmWRKY44表达模式。克隆了一个水曲柳WKRY基因,编码区长1 383 bp,编码460氨基酸。对其编码蛋白分析发现其为稳定亲水蛋白,亚细胞定位预测主要在细胞核,进行保守域及同源性分析分析,属于WRKYⅠ类家族,命名为FmWRKY44。qRT-PCR分析发现,FmWRKY44在种子中高度表达,并不同程度响应低温、高温、盐和干旱4种非生物胁迫。同时发现FmWRKY44与NAA、ABA、GA_3、JA植物激素响应,水曲柳FmWRKY44基因积极响应低温、高温胁迫。
To clone the FmWRKY44 gene from Fraxinus mandshurica and explore its role in abiotic stress and hormone stress,specific primers were designed using the sequences of the variceal drought-transcriptome,the complete ORF sequence of FmWRKY44 gene was cloned,and bioinformatics analysis was carried out on the sequence and its coded products. The expression pattern of FmWRKY44 was analyzed using qRT-PCR. A WKRY gene of F. mandshurica was cloned,encoding 1 383 bp,encoding 460 amino acid. It was found to be a stable hydrophilic protein by the analysis of its encoded protein. The subcellular localization was mainly predicted in the nucleus,and the conserved domain and homology were analyzed. It belonged to the WRKY class I family and was named FmWRKY44. By qRT-PCR analysis,FmWRKY44 was highly expressed in the seeds. Different degrees of response to low temperature,high temperature,salt and drought four abiotic stresses. FmWRKY44 and NAA,ABA,GA_3,JA phytohormone responses were also found. FmWRKY44 gene of F. mandshurica positively responds to low temperature and high temperature stress.
To clone the FmWRKY44 gene from Fraxinus mandshurica and explore its role in abiotic stress and hormone stress,specific primers were designed using the sequences of the variceal drought-transcriptome,the complete ORF sequence of FmWRKY44 gene was cloned,and bioinformatics analysis was carried out on the sequence and its coded products. The expression pattern of FmWRKY44 was analyzed using qRT-PCR. A WKRY gene of F. mandshurica was cloned,encoding 1 383 bp,encoding 460 amino acid. It was found to be a stable hydrophilic protein by the analysis of its encoded protein. The subcellular localization was mainly predicted in the nucleus,and the conserved domain and homology were analyzed. It belonged to the WRKY class I family and was named FmWRKY44. By qRT-PCR analysis,FmWRKY44 was highly expressed in the seeds. Different degrees of response to low temperature,high temperature,salt and drought four abiotic stresses. FmWRKY44 and NAA,ABA,GA_3,JA phytohormone responses were also found. FmWRKY44 gene of F. mandshurica positively responds to low temperature and high temperature stress.
引文
1. Liu X T,Cao X Q,Shi S C,et al. Comparative RNA-Seq analysis reveals a critical role for brassinosteroids in rose(Rosa hybrida)petal defense against Botrytis cinerea infection[J]. BMC Genet,2018,19:62.
2 .郑超,郑二松,王栩鸣,等.水稻WRKY转录调控因子研究进展[J].生物技术通讯,2018,29(2):286-294.Zheng C,Zheng E S,Wang X M,et al. Research progress on rice WRKY transcription factors[J]. Letters in Biotechnology,2018,29(2):286-294.
3 . Rushton P J,Torres J T,Parniske M,et al. Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes[J]. EMBO Journal,1996,15(20):5690-5700.
4 . Liu J J,Xiang Y. In silico mining and PCR-based approaches to transcription factor discovery in non-model plants:gene discovery of the WRKY transcription factors in conifers[J]. Methods in Molecular Biology,2011,754:21-43.
5 . Li H E,Xu Y,Xiao Y,et al. Expression and functional analysis of two genes encoding transcription factors,VpWRKY1 and Vp WRKY2,isolated from Chinese wild Vitis pseudoreticulata[J]. Planta,2010,232(6):1325-1337.
6 . Zhang Y J,Wang L J. The WRKY transcription factor superfamily:its origin in eukaryotes and expansion in plants[J]. BMC Evolutionary Biology,2005,5:1.
7 . Eulgem T,Rushton P J,Robatzek S,et al. The WRKY superfamily of plant transcription factors[J]. Trends in Plant Science,2000,5(5):199-206.
8 .李珍,华秀婷,张积森.高等植物WRKY转录因子家族的演化及功能研究进展[J].热带作物学报,2018,39(2):405-414.Li Z,Hua X T,Zhang J S. Evolution and gene function of WRKY transcription factor families in higher plants[J].Chinese Journal of Tropical Crops,2018,39(2):405-414.
9 . Wang C T,Ru J N,Liu Y W,et al. Maize WRKY transcription factor Zm WRKY106 confers drought and heat tolerance in transgenic plants[J]. International Journal of Molecular Sciences,2018,19(10):3046.
10 . He L,Wu Y H,Zhao Q,et al. Chrysanthemum Dg WRKY2gene enhances tolerance to salt stress in transgenic chrysanthemum[J]. International Journal of Molecular Sciences,2018,19(7):2062.
11 . Bao W Q,Wang X W,Chen M,et al. A WRKY transcription factor,Pc WRKY33,from Polygonum cuspidatum reduces salt tolerance in transgenic Arabidopsis thaliana[J].Plant Cell Reports,2018,37(7):1033-1048.
12 . Kim C Y,Vo K T X,Nguyen C D,et al. Functional analysis of a cold-responsive rice WRKY gene,Os WRKY71[J].Plant Biotechnology Reports,2016,10(1):13-23.
13 .丛庆,张琪,宋丽莉,等.激素在植物冷胁迫应答中的角色[J].核农学报,2016,30(3):614-619.Cong Q,Zhang Q,Song L L,et al. Roles of phytohormones on cold response in plants[J]. Journal of Nuclear Agricultural Sciences,2016,30(3):614-619.
14 . Zhang Y,Yu H J,Yang X Y,et al. Cs WRKY46,a WRKY transcription factor from cucumber,confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an ABA-dependent manner[J]. Plant Physiology and Biochemistry,2016,108:478-487.
15 .孙丽敏,裴雁曦,刘志强. H2S信号和WRKY在ABA调节根生长和气孔运动中的关系[J].植物研究,2016,36(1):97-104.Sun L M,Pei Y X,Liu Z Q. Relationship between H2S signal and WRKY in ABA regulating root growth and stomatal movement[J]. Bulletin of Botanical Research,2016,36(1):97-104.
16 . Li S J,Zhou X,Chen L G,et al. Functional characterization of Arabidopsis thaliana WRKY39 in heat stress[J]. Molecules and Cells,2010,29(5):475-483.
17 . Zhang L Y,Gu L K,Ringler P,et al. Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells[J]. Plant Science,2015,236:214-222.
18 . Zhang L P,Chen L G,Yu D Q. Transcription factor WRKY75 interacts with DELLA proteins to affect flowering[J]. Plant Physiology,2018,176(1):790-803.
19 .陆爱君,曾凡顺,冯健,等.我国水曲柳育苗技术研究进展[J].辽宁林业科技,2015,(3):43-45,76.Lu A J,Zeng F S,Feng J,et al. Research progress on mulberry seedling technology in China[J]. Liaoning Forestry Science and Technology,2015,(3):43-45,76.
20 .刘颖,何利明,赵兴堂,等.水曲柳WRKY40基因的克隆及表达模式分析[J].分子植物育种,2017,15(3):833-838.Liu Y,He L M,Zhao X T,et al. Cloning and expression pattern analysis of Fm WRKY40 gene in Fraxinus mandshurica[J]. Molecular Plant Breeding,2017,15(3):833-838.
21 . Li D H,Liu P,Yu J Y,et al. Genome-wide analysis of WRKY gene family in the sesame genome and identification of the WRKY genes involved in responses to abiotic stresses[J]. BMC Plant Biology,2017,17:152.
22 .郝林,徐昕.植物转录因子WRKY家族的结构及功能[J].植物生理学通讯,2004,40(2):260-265.Hao L,Xu X. The structure and function of WRKY superfamily of plant transcription factors[J]. Plant Physiology Communications,2004,40(2):260-265.
23 . Finatto T,Viana V E,Woyann L G,et al. Can WRKY transcription factors help plants to overcome environmental challenges?[J]. Genetics and Molecular Biology,2018,41(3):533-544.
24 . De Zelicourt A,Colcombet J,Hirt H. The role of MAPK modules and aba during abiotic stress signaling[J]. Trends in Plant Science,2016,21(8):677-685.
25 . Jiang J J,Ma S H,Ye N H,et al. WRKY transcription factors in plant responses to stresses[J]. Journal of Integrative Plant Biology,2017,59(2):86-101.
26 . Sheikh A H,Eschen-Lippold L,Pecher P,et al. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana[J]. Frontiers in Plant Science,2016,7:61.
27 .孙晓梅. MAPK-WRKY模块调节质体反向信号的研究[D].上海:上海师范大学,2018.Sun X M. Study on MAPK-WRKY module regulates plastid retrograde signaling[D]. Shanghai:Shanghai Normal University,2018.
28 .王娜,张振葆,黄凤珠,等. WRKY转录因子参与植物非生物胁迫应答的研究进展[J].核农学报,2014,28(10):1819-1827.Wang N,Zhang Z B,Huang F Z,et al. Advance on the role of WRKY transcription factors in abiotic stress response[J]. Journal of Nuclear Agricultural Sciences,2014,28(10):1819-1827.
29 . Geilen K,Heilmann M,Hillmer S,et al. WRKY43 regulates polyunsaturated fatty acid content and seed germination under unfavourable growth conditions[J]. Scientific Reports,2017,7(1):14235.
30 . Wang C T,Ru J N,Liu Y W,et al. The maize WRKY transcription factor Zm WRKY40 confers drought resistance in transgenic Arabidopsis[J]. International Journal of Molecular Sciences,2018,19(9):2580.
31 . Cheng M N,Huang Z J,Hua Q Z,et al. The WRKY transcription factor Hp WRKY44 regulates Cyt P450-like1 expression in red pitaya fruit(Hylocereus polyrhizus)[J].Horticulture Research,2017,4:17039.
2 .郑超,郑二松,王栩鸣,等.水稻WRKY转录调控因子研究进展[J].生物技术通讯,2018,29(2):286-294.Zheng C,Zheng E S,Wang X M,et al. Research progress on rice WRKY transcription factors[J]. Letters in Biotechnology,2018,29(2):286-294.
3 . Rushton P J,Torres J T,Parniske M,et al. Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes[J]. EMBO Journal,1996,15(20):5690-5700.
4 . Liu J J,Xiang Y. In silico mining and PCR-based approaches to transcription factor discovery in non-model plants:gene discovery of the WRKY transcription factors in conifers[J]. Methods in Molecular Biology,2011,754:21-43.
5 . Li H E,Xu Y,Xiao Y,et al. Expression and functional analysis of two genes encoding transcription factors,VpWRKY1 and Vp WRKY2,isolated from Chinese wild Vitis pseudoreticulata[J]. Planta,2010,232(6):1325-1337.
6 . Zhang Y J,Wang L J. The WRKY transcription factor superfamily:its origin in eukaryotes and expansion in plants[J]. BMC Evolutionary Biology,2005,5:1.
7 . Eulgem T,Rushton P J,Robatzek S,et al. The WRKY superfamily of plant transcription factors[J]. Trends in Plant Science,2000,5(5):199-206.
8 .李珍,华秀婷,张积森.高等植物WRKY转录因子家族的演化及功能研究进展[J].热带作物学报,2018,39(2):405-414.Li Z,Hua X T,Zhang J S. Evolution and gene function of WRKY transcription factor families in higher plants[J].Chinese Journal of Tropical Crops,2018,39(2):405-414.
9 . Wang C T,Ru J N,Liu Y W,et al. Maize WRKY transcription factor Zm WRKY106 confers drought and heat tolerance in transgenic plants[J]. International Journal of Molecular Sciences,2018,19(10):3046.
10 . He L,Wu Y H,Zhao Q,et al. Chrysanthemum Dg WRKY2gene enhances tolerance to salt stress in transgenic chrysanthemum[J]. International Journal of Molecular Sciences,2018,19(7):2062.
11 . Bao W Q,Wang X W,Chen M,et al. A WRKY transcription factor,Pc WRKY33,from Polygonum cuspidatum reduces salt tolerance in transgenic Arabidopsis thaliana[J].Plant Cell Reports,2018,37(7):1033-1048.
12 . Kim C Y,Vo K T X,Nguyen C D,et al. Functional analysis of a cold-responsive rice WRKY gene,Os WRKY71[J].Plant Biotechnology Reports,2016,10(1):13-23.
13 .丛庆,张琪,宋丽莉,等.激素在植物冷胁迫应答中的角色[J].核农学报,2016,30(3):614-619.Cong Q,Zhang Q,Song L L,et al. Roles of phytohormones on cold response in plants[J]. Journal of Nuclear Agricultural Sciences,2016,30(3):614-619.
14 . Zhang Y,Yu H J,Yang X Y,et al. Cs WRKY46,a WRKY transcription factor from cucumber,confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an ABA-dependent manner[J]. Plant Physiology and Biochemistry,2016,108:478-487.
15 .孙丽敏,裴雁曦,刘志强. H2S信号和WRKY在ABA调节根生长和气孔运动中的关系[J].植物研究,2016,36(1):97-104.Sun L M,Pei Y X,Liu Z Q. Relationship between H2S signal and WRKY in ABA regulating root growth and stomatal movement[J]. Bulletin of Botanical Research,2016,36(1):97-104.
16 . Li S J,Zhou X,Chen L G,et al. Functional characterization of Arabidopsis thaliana WRKY39 in heat stress[J]. Molecules and Cells,2010,29(5):475-483.
17 . Zhang L Y,Gu L K,Ringler P,et al. Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells[J]. Plant Science,2015,236:214-222.
18 . Zhang L P,Chen L G,Yu D Q. Transcription factor WRKY75 interacts with DELLA proteins to affect flowering[J]. Plant Physiology,2018,176(1):790-803.
19 .陆爱君,曾凡顺,冯健,等.我国水曲柳育苗技术研究进展[J].辽宁林业科技,2015,(3):43-45,76.Lu A J,Zeng F S,Feng J,et al. Research progress on mulberry seedling technology in China[J]. Liaoning Forestry Science and Technology,2015,(3):43-45,76.
20 .刘颖,何利明,赵兴堂,等.水曲柳WRKY40基因的克隆及表达模式分析[J].分子植物育种,2017,15(3):833-838.Liu Y,He L M,Zhao X T,et al. Cloning and expression pattern analysis of Fm WRKY40 gene in Fraxinus mandshurica[J]. Molecular Plant Breeding,2017,15(3):833-838.
21 . Li D H,Liu P,Yu J Y,et al. Genome-wide analysis of WRKY gene family in the sesame genome and identification of the WRKY genes involved in responses to abiotic stresses[J]. BMC Plant Biology,2017,17:152.
22 .郝林,徐昕.植物转录因子WRKY家族的结构及功能[J].植物生理学通讯,2004,40(2):260-265.Hao L,Xu X. The structure and function of WRKY superfamily of plant transcription factors[J]. Plant Physiology Communications,2004,40(2):260-265.
23 . Finatto T,Viana V E,Woyann L G,et al. Can WRKY transcription factors help plants to overcome environmental challenges?[J]. Genetics and Molecular Biology,2018,41(3):533-544.
24 . De Zelicourt A,Colcombet J,Hirt H. The role of MAPK modules and aba during abiotic stress signaling[J]. Trends in Plant Science,2016,21(8):677-685.
25 . Jiang J J,Ma S H,Ye N H,et al. WRKY transcription factors in plant responses to stresses[J]. Journal of Integrative Plant Biology,2017,59(2):86-101.
26 . Sheikh A H,Eschen-Lippold L,Pecher P,et al. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana[J]. Frontiers in Plant Science,2016,7:61.
27 .孙晓梅. MAPK-WRKY模块调节质体反向信号的研究[D].上海:上海师范大学,2018.Sun X M. Study on MAPK-WRKY module regulates plastid retrograde signaling[D]. Shanghai:Shanghai Normal University,2018.
28 .王娜,张振葆,黄凤珠,等. WRKY转录因子参与植物非生物胁迫应答的研究进展[J].核农学报,2014,28(10):1819-1827.Wang N,Zhang Z B,Huang F Z,et al. Advance on the role of WRKY transcription factors in abiotic stress response[J]. Journal of Nuclear Agricultural Sciences,2014,28(10):1819-1827.
29 . Geilen K,Heilmann M,Hillmer S,et al. WRKY43 regulates polyunsaturated fatty acid content and seed germination under unfavourable growth conditions[J]. Scientific Reports,2017,7(1):14235.
30 . Wang C T,Ru J N,Liu Y W,et al. The maize WRKY transcription factor Zm WRKY40 confers drought resistance in transgenic Arabidopsis[J]. International Journal of Molecular Sciences,2018,19(9):2580.
31 . Cheng M N,Huang Z J,Hua Q Z,et al. The WRKY transcription factor Hp WRKY44 regulates Cyt P450-like1 expression in red pitaya fruit(Hylocereus polyrhizus)[J].Horticulture Research,2017,4:17039.