海岛棉GbAGL15和GbAIL5的克隆及其在低温诱导XH16体胚发生同步化过程中的表达
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摘要
体细胞胚胎发生的同步化有助于加快作物分子育种的进程,体胚发生过程中Agamous-like15(AGL15)、Aintegumenta-like5 (AIL5)等关键基因特异性表达起着类似开关的作用。为了进一步探明低温诱导体细胞胚胎同步化发生的分子机理,本研究利用同源序列从海岛棉(Gossypium barbadense) XH16胚性愈伤组织中克隆得到GbAGL15 (GenBank No. MK580460)和GbAIL5 (GenBank No. MK591943)核苷酸序列,并利用qRT-PCR技术研究低温处理后胚性细胞中GbAGL15和GbAIL5基因的表达模式。结果表明,GbAGL15基因编码区全长为753 bp,编码250个氨基酸,含有MADS-MEF2-like结构域和K-box保守区,与雷蒙德氏棉(G. raimondii) GrAGL15、亚洲棉(G. arboretum) GaAGL15亲缘关系较近;GbAIL5基因编码区全长为1 626 bp,编码541个氨基酸,含有2个AP2结构域,与陆地棉(G. hirsutum) GhAIL5在同一进化分支上。qRT-PCR显示,在胚性愈伤组织低温处理后GbAGL15基因与对照组相比表达量极显著的降低(P<0.01),GbAIL5基因的表达量呈现缓慢下降的趋势,推测GbAGL15和GbAIL5基因参与体细胞胚胎发生并起调控作用,在低温条件下其基因表达被抑制,致使低温处理下胚性愈伤组织发育分化停滞,从而促进体胚的同步化发生。本研究为揭示海岛棉体细胞同步化发生的分子机制提供了理论依据,有助于海岛棉体细胞胚发生的遗传转化体系的建立。
Synchronization of somatic embryogenesis helps accelerate the process of crop molecular breeding. The specific expression of key genes such as Agamous-like15(AGL15) and Aintegumenta-like5(AIL5) plays a role similar to switch during somatic embryogenesis. To further explore the molecular mechanism of hypothermia induced somatic embryo synchronization, in this study, GbAGL15(GenBank No.MK580460) and GbAIL5(GenBank No. MK591943) were cloned by homologous sequences methods from the embryonic callus of Gossypium barbadense XH16, and the expression pattern of GbAGL15 and GbAIL5 genes in low temperature treated embryogenic cells were checked by qRT-PCR. The results showed that the coding region of GbAGL15 gene was 753 bp and encoded 250 amino acids, which contained the MADS-MEF2-like domain and the K-box conserved region. Phylogenetic tree analysis showed that there was a close relationship among GbAGL15 together with GrAGL15(G. raimondii) and GaAGL15(G. arboretum). The coding region of GbAIL5 gene was 1 626 bp and encoded 541 amino acid in the coding segment, and it contained two AP2 domains. Phylogenetic tree analysis showed that GbAIL5 was located in the same evolutionary branch with GhAIL5(G. hirsutum). qRT-PCR results showed that the expression levels of GbAGL15 and GbAIL5 in embryogenic callus were significantly lower than those of the control group after low temperature treatment. It was speculated that the GbAGL15 and GbAIL5 genes might be involved in somatic embryogenesis and played regulatory roles. Under low temperature conditions, the two gene expression might be inhibited, resulting in the development and differentiation of hypocotyl callus tissue at low temperature, which might promotes the synchronization of somatic embryos. And after the stress conditions were removed, the embryo development could be swithced on in synchronization pattern. The study partially elucidates the mechanism of hypothermia induced somatic embryo synchronization, promoting the foundation of high efficient system of somatic embryogenesis-dependent genetic transformation in G. barbadense.
Synchronization of somatic embryogenesis helps accelerate the process of crop molecular breeding. The specific expression of key genes such as Agamous-like15(AGL15) and Aintegumenta-like5(AIL5) plays a role similar to switch during somatic embryogenesis. To further explore the molecular mechanism of hypothermia induced somatic embryo synchronization, in this study, GbAGL15(GenBank No.MK580460) and GbAIL5(GenBank No. MK591943) were cloned by homologous sequences methods from the embryonic callus of Gossypium barbadense XH16, and the expression pattern of GbAGL15 and GbAIL5 genes in low temperature treated embryogenic cells were checked by qRT-PCR. The results showed that the coding region of GbAGL15 gene was 753 bp and encoded 250 amino acids, which contained the MADS-MEF2-like domain and the K-box conserved region. Phylogenetic tree analysis showed that there was a close relationship among GbAGL15 together with GrAGL15(G. raimondii) and GaAGL15(G. arboretum). The coding region of GbAIL5 gene was 1 626 bp and encoded 541 amino acid in the coding segment, and it contained two AP2 domains. Phylogenetic tree analysis showed that GbAIL5 was located in the same evolutionary branch with GhAIL5(G. hirsutum). qRT-PCR results showed that the expression levels of GbAGL15 and GbAIL5 in embryogenic callus were significantly lower than those of the control group after low temperature treatment. It was speculated that the GbAGL15 and GbAIL5 genes might be involved in somatic embryogenesis and played regulatory roles. Under low temperature conditions, the two gene expression might be inhibited, resulting in the development and differentiation of hypocotyl callus tissue at low temperature, which might promotes the synchronization of somatic embryos. And after the stress conditions were removed, the embryo development could be swithced on in synchronization pattern. The study partially elucidates the mechanism of hypothermia induced somatic embryo synchronization, promoting the foundation of high efficient system of somatic embryogenesis-dependent genetic transformation in G. barbadense.
引文
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王哲之,张大力.1990.陆地棉愈伤组织产生及胚胎发生的细胞学研究[J].西北植物学报,10(2):77-83.(Wang ZZ,Zhang D L.1990.Cat cytological observation on callus production and embryogenesis of Gossypium hirsutum L[J].Acta Botanica Boreali-Occidentalia Sinica,10(2):77-83.)
张宇,沈海龙.2007.植物体细胞胚同步化发生的控制[J].植物生理学通讯,(3):583-587.(Zhang Y,Shen H L.2007.Control of synchronization for plant somatic embryogenesis[J].Plant Physiology Communications,(3):583-587)
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Harding E W,Tang W,Nichols K W,et al.2003.Expression and maintenance of embryogenic potential is enhanced through constitutive expression of Agamous-like 15[J].Plant Physiology,133(2):653-663.
Jofuku K D,Omidyar P K,Gee Z,et al.2005.Control of seed mass and seed yield by the floral homeotic gene APETALA2[J].Proceedings of the National Academy of Sciences of the USA,102(8):3117-3122.
Ouakfaoui S E,Schnell J,Abdeen A,et al.2010.Control of somatic embryogenesis and embryo development by AP2 transcription factors[J].Plant Molecular Biology,74(4-5):313-326.
Quiroz-Figueroa F R,Rojas-Herrear R,Galaz-Avalos R M.2006.Embryo production through somatic embryogenesis can be used to study cell differentiation in plants[J].Plant Cell,Tissue and Organ Culture,86(3):285-301.
Nole-Wilson S,Tranby T L,Krizek B A.2005.Aintegumentalike(AIL)genes are expressed in young tissues and may specify meristematic or division-competent states[J].Plant Molecular Biology,57(5):613-28.
Tsuwamoto R,Yokoi S,Takahata Y.2010.Arabidopsis EM-BRYOMAKER encoding an AP2 domain transcription factor plays a key role in developmental change from vegetative to embryonic phase[J].Plant Molecular Biology,73:481-492.
Wang H,Caruso L V,Downie A B,et al.2004.The embryo MADS domain protein Agamous-Like 15 directly regulates expression of a gene encoding an enzyme involved in gibberellin metabolism[J].The Plant Cell,16(5):1206-1219.
Yang Z,Li C,Wang Y,et al.2014.GhAGL15s,preferentially expressed during somatic embryogenesis,promote embryogenic callus formation in Cotton(Gossypium hirsutum L.)[J].Molecular Genetics and Genomics,289(5):873-883.
Zheng Q L,Zheng Y M,Perry S E,et al.2013.Decreased GmAGL15 expression and reduced ethylene synthesis may contribute to reduced somatic embryogenesis in a poorly embryogenic cultivar of glycine max[J].Plant Signaling and Behavior,8(9):e25422.
Zheng Q,Perry S E.2014.Alterations in the transcriptome of soybean in response to enhanced somatic embryogenesis promoted by orthologs of Agamous-like15 and Agamous-like18[J].Plant Physiology,164(3):1365-1377.
Zheng Q,Zheng Y,Ji H,et al.2016.Gene regulation by the AGL15 transcription factor reveals hormone interactions in somatic embryogenesis[J].Plant Physiology,172(4):2374-2387.
郭家雁,张霞,丁喜莲,等.2017.不同处理诱导XH16号体细胞胚胎同步化发生[J].棉花学报,29(4):385-392.(Guo J Y,Zhang X,Ding X L,et al.2017.Synchronized somatic embryogenesis of"Xinhai 16"induced by different treatments[J].Cotton Science,29(4):385-392.)
姜福星,魏丕伟,寇亚平,等.2017.探析植物体胚发生的关键基因[J].分子植物育种,4:1304-1311.(Jiang F X,Wei P W,Kou Y P,et al.2017.Evaluation and analysis of key genes in plant somatic embryogenesis[J].Molecular Plant Breeding,4:1304-1311.)
毛春娜,张爱民,薛建平,等.2011.低温处理对半夏悬浮培养细胞同步化的影响[J].中国中药杂志,36(8):959-962.(Mao C N,Zhang A M,Xue J P,et al.2011.Effect of low temperature on cell synchronization division in suspension cultore cells of Pinellia ternata[J].China Journal of Chinese Materia Medica,36(8):959-962.)
梅兴国,周爱文.2001.红豆杉细胞同步化的研究[J].生物技术,11(3):9-10.(Mei X G,Zhou A W.2001.Studies on synchronization of taxus cells[J].Biotechnology,11(3):9-10.)
王兴春,李宏,王敏,等.2010.植物体细胞胚胎发生的调控网络[J].生物工程学报,26(2):141-146.(Wang X C,Li H,Wang M,et al.2010.Regulatory networks of somatic embryogenesis in plant[J].Chinese Journal of Biotechnology,26(2):141-146.)
王哲之,张大力.1990.陆地棉愈伤组织产生及胚胎发生的细胞学研究[J].西北植物学报,10(2):77-83.(Wang ZZ,Zhang D L.1990.Cat cytological observation on callus production and embryogenesis of Gossypium hirsutum L[J].Acta Botanica Boreali-Occidentalia Sinica,10(2):77-83.)
张宇,沈海龙.2007.植物体细胞胚同步化发生的控制[J].植物生理学通讯,(3):583-587.(Zhang Y,Shen H L.2007.Control of synchronization for plant somatic embryogenesis[J].Plant Physiology Communications,(3):583-587)
Ashish A,Ganesan P,Gary B,et al.2009.Identification and analysis of transcription factor family-specific features derived from DNA and protein information[J].Pattern Recognition Letters,31(14):2097-2102.
Fernandez D E,Heck G R,Perry S E,et al.2000.The embryo MADS domain factor AGL15 acts postembryonically:Inhibition of perianth senescence and abscission via constitutive expression[J].The Plant Cell,12(2):183-197.
Harding E W,Tang W,Nichols K W,et al.2003.Expression and maintenance of embryogenic potential is enhanced through constitutive expression of Agamous-like 15[J].Plant Physiology,133(2):653-663.
Jofuku K D,Omidyar P K,Gee Z,et al.2005.Control of seed mass and seed yield by the floral homeotic gene APETALA2[J].Proceedings of the National Academy of Sciences of the USA,102(8):3117-3122.
Ouakfaoui S E,Schnell J,Abdeen A,et al.2010.Control of somatic embryogenesis and embryo development by AP2 transcription factors[J].Plant Molecular Biology,74(4-5):313-326.
Quiroz-Figueroa F R,Rojas-Herrear R,Galaz-Avalos R M.2006.Embryo production through somatic embryogenesis can be used to study cell differentiation in plants[J].Plant Cell,Tissue and Organ Culture,86(3):285-301.
Nole-Wilson S,Tranby T L,Krizek B A.2005.Aintegumentalike(AIL)genes are expressed in young tissues and may specify meristematic or division-competent states[J].Plant Molecular Biology,57(5):613-28.
Tsuwamoto R,Yokoi S,Takahata Y.2010.Arabidopsis EM-BRYOMAKER encoding an AP2 domain transcription factor plays a key role in developmental change from vegetative to embryonic phase[J].Plant Molecular Biology,73:481-492.
Wang H,Caruso L V,Downie A B,et al.2004.The embryo MADS domain protein Agamous-Like 15 directly regulates expression of a gene encoding an enzyme involved in gibberellin metabolism[J].The Plant Cell,16(5):1206-1219.
Yang Z,Li C,Wang Y,et al.2014.GhAGL15s,preferentially expressed during somatic embryogenesis,promote embryogenic callus formation in Cotton(Gossypium hirsutum L.)[J].Molecular Genetics and Genomics,289(5):873-883.
Zheng Q L,Zheng Y M,Perry S E,et al.2013.Decreased GmAGL15 expression and reduced ethylene synthesis may contribute to reduced somatic embryogenesis in a poorly embryogenic cultivar of glycine max[J].Plant Signaling and Behavior,8(9):e25422.
Zheng Q,Perry S E.2014.Alterations in the transcriptome of soybean in response to enhanced somatic embryogenesis promoted by orthologs of Agamous-like15 and Agamous-like18[J].Plant Physiology,164(3):1365-1377.
Zheng Q,Zheng Y,Ji H,et al.2016.Gene regulation by the AGL15 transcription factor reveals hormone interactions in somatic embryogenesis[J].Plant Physiology,172(4):2374-2387.