茶树生长素合成酶基因YUCCA10克隆与表达分析
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摘要
【目的】探究YUCCA10基因在正常花和不育花中的表达规律,为深入探索YUCCA10基因和生长素合成对茶树花器官发育的调控机制提供理论基础。【方法】前期研究茶树正常花与不育花转录组差异时,获得一条与YUCCA10基因高度同源的基因序列,利用‘云茶1号'茶树全长转录组数据库以及PCR技术克隆验证茶树YUCCA10基因,并对其生物信息学特征进行分析,同时运用qRT-PCR技术研究其表达特征。【结果】经验证YUCCA10基因含有1个1137 bp的开放阅读框,编码379个氨基酸,分子量为42.63 kDa,等电点为8.88,茶树YUCCA10蛋白具有NADPH结合位点和FAD结合位点,与多种植物YUCCA10蛋白同源。qRT-PCR分析CsYUCCA10在正常花中随着花的生长发育表达量增加,而在不育花的花瓣、雄蕊、雌蕊以及不同发育期中的表达均低于正常花。【结论】茶树CsYUCCA10基因在不育花中不能正常合成,从而影响了花的发育导致不育。
【Objective】 In order to explore the molecular mechanism of YUCCA10 gene and auxin synthesis on the development of tea plant flower, the expression pattern of YUCCA10 gene was studied in normal flowers and sterile flowers. 【Method】 In the early study of the difference between normal and sterile flower transcriptome of tea plant, a gene sequence highly homologous to YUCCA10 gene was obtained. The full-length transcriptome database of ‘Yuncha 1' tea plant and PCR technology were used to clone and verify the YUCCA10 gene, and its bioinformatics characteristics were analyzed. Meanwhile, qRT-PCR technology was applied to study its expression characteristics. 【Result】 It was proved that the YUCCA10 gene contained one open reading frame of 1137 bp, encoding 379 amino acids, molecular weight of 42.63 kDa, and the isoelectric point of 8.88. Tea plant YUCCA10 protein had the binding sites of NADPH and FAD, which was homologous to various plants' YUCCA10 proteins. CsYUCCA10 expression by QRT-PCR analysis in normal flowers increased with the growth and development of flowers, while the expressions in petals, stamens, pistils and different developmental periods of sterile flowers were lower than that in normal flowers. 【Conclusion】 The CsYUCCA10 gene of tea plant could not be synthesized normally in sterile flowers, which affected the development of flowers and led to infertility.
【Objective】 In order to explore the molecular mechanism of YUCCA10 gene and auxin synthesis on the development of tea plant flower, the expression pattern of YUCCA10 gene was studied in normal flowers and sterile flowers. 【Method】 In the early study of the difference between normal and sterile flower transcriptome of tea plant, a gene sequence highly homologous to YUCCA10 gene was obtained. The full-length transcriptome database of ‘Yuncha 1' tea plant and PCR technology were used to clone and verify the YUCCA10 gene, and its bioinformatics characteristics were analyzed. Meanwhile, qRT-PCR technology was applied to study its expression characteristics. 【Result】 It was proved that the YUCCA10 gene contained one open reading frame of 1137 bp, encoding 379 amino acids, molecular weight of 42.63 kDa, and the isoelectric point of 8.88. Tea plant YUCCA10 protein had the binding sites of NADPH and FAD, which was homologous to various plants' YUCCA10 proteins. CsYUCCA10 expression by QRT-PCR analysis in normal flowers increased with the growth and development of flowers, while the expressions in petals, stamens, pistils and different developmental periods of sterile flowers were lower than that in normal flowers. 【Conclusion】 The CsYUCCA10 gene of tea plant could not be synthesized normally in sterile flowers, which affected the development of flowers and led to infertility.
引文
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[14]宋维希,孙云南,刘德和,等.回交不育茶树花及其亲本的生化特性比较分析[J].南方农业学报, 2017, 48:11-13.
[15]Dai X H, Mashiguchi K, Chen Q G, et al. The biochemical mechanism of auxin biosynthesis by an Arabidopsis YUCCA Flavin-containing Monooxygenase[J]. J Bio Chem, 2013, 288: 1448-1457.
[16]Pagnussat G C, Alandete-Saez M, Bowman J L, et al. Auxin-dependent patterning and gamete specifi cation in the Arabidopsis female gametophyte[J]. Science, 2009,324: 1684-1689.
[17]McSteen P. Auxin and monocot development[J]. Cold Spring Harb Perspect Biol, 2010(2): a001479.
[18]Zhao Y D. Auxin biosynthesis: a simple two-step pathway converts tryptophan to indole-3-acetic acid in plants[J]. Mol Plant, 2012(5): 334-338.
[19]阚东阳,柯学, Walid Ghidan,等.拟南芥图位克隆快速初定位系统的建立[J].西南农业学报, 2018, 31(9):1765-1771.
[20]李娜.小麦TaYUC基因的克隆与初步功能分析(学位论文)[D].青岛:山东农业大学,2014.
[21]Di D W, Wu L, Zhang L, et al. Functional roles of Arabidopsis CKRC2/YUCCA8 gene and the involvement of PIF4 in the regulation of auxin biosynthesis by cytokinin[J]. Scientific Reports, 2016(6):36-66.
[22]Woodward A W, Bartel B. Auxin: regulation, action, and interaction[J]. Ann Bot, 2005, 95: 707-735.
[23]Vanneste S, Friml J. Auxin: a trigger for change in plant development[J]. Cell, 2009,136: 1005-1016.
[2]Cheng Y F, Dai X H, Zhao Y D. Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis[J]. Genes & De, 2006, 20: 1790-1799.
[3]Cheng Y F, Dai X H, Zhao Y D. Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis[J]. Plant Cell, 2007,19: 2430-2439.
[4]Mashiguchia K, Tanaka K, Sakaic T, et al. The main auxin biosynthesis pathway in Arabidopsis[J]. Proc Natl Acad Sci USA, 2011,108: 18512-18517.
[5]Won C, Shen X, Mashiguchi K, et al. Conversion of tryptophan to indole-3-acetic acid by Trypto-phan aminotransferases of Arabidopsis and YUCCAs in Arabidopsis[J]. Proc Natl Acad Sci USA, 2011,108: 18518-18523.
[6]Axel P, Verena K. Bioinformatics analysis of phylogeny and transcription of TAA/YUC auxin biosynthetic genes[J]. Int J Mol Sci, 2017,18: 1791: 2-17.
[7]Kriechbaumer V, Botchway S W, Hawes C. Localization and interactions between Arabidopsis auxin biosynthetic enzymes in the TAA/YUC-dependent pathway[J]. J Exp Bot, 2016,67: 4195-4207.
[8]Zhao Y D. Auxin biosynthesis and its role in plant development[J]. Annu Rev Plant Biol, 2010, 61: 49-64.
[9]Liu H, Xie WF, Zhang L, et al. Auxin biosynthesis by the YUCCA6 flavin monooxygenase gene in woodland strawberry[J]. J Integr Plant Biol, 2014, 56: 350-363.
[10]Adams K R, Johnson K L, Murphy T M. Prehistoric Puebloan yucca (Yucca) quids with wild tobacco (Nicotiana) contents: Molecular and morphological evidence from Antelope Cave, northwestern Arizona[J]. J Fie Arch, 2015, 40: 310-324.
[11]Yamamoto Y, Kamiya N, Morinaka Y, et al. Auxin biosynthesis by the YUCCA genes in rice[J]. Plant Physiol, 2007,143: 1362-1371.
[12]陈林波,夏丽飞,田易萍,等.基于数字基因表达谱分析的茶树花不育基因挖掘[J].作物学报, 2017,43: 210-217.
[13]朱兴正,夏丽飞,陈林波,等.保护品种云茶1号茶树全长转录组测序分析[J].茶叶科学, 2018,38: 193-201.
[14]宋维希,孙云南,刘德和,等.回交不育茶树花及其亲本的生化特性比较分析[J].南方农业学报, 2017, 48:11-13.
[15]Dai X H, Mashiguchi K, Chen Q G, et al. The biochemical mechanism of auxin biosynthesis by an Arabidopsis YUCCA Flavin-containing Monooxygenase[J]. J Bio Chem, 2013, 288: 1448-1457.
[16]Pagnussat G C, Alandete-Saez M, Bowman J L, et al. Auxin-dependent patterning and gamete specifi cation in the Arabidopsis female gametophyte[J]. Science, 2009,324: 1684-1689.
[17]McSteen P. Auxin and monocot development[J]. Cold Spring Harb Perspect Biol, 2010(2): a001479.
[18]Zhao Y D. Auxin biosynthesis: a simple two-step pathway converts tryptophan to indole-3-acetic acid in plants[J]. Mol Plant, 2012(5): 334-338.
[19]阚东阳,柯学, Walid Ghidan,等.拟南芥图位克隆快速初定位系统的建立[J].西南农业学报, 2018, 31(9):1765-1771.
[20]李娜.小麦TaYUC基因的克隆与初步功能分析(学位论文)[D].青岛:山东农业大学,2014.
[21]Di D W, Wu L, Zhang L, et al. Functional roles of Arabidopsis CKRC2/YUCCA8 gene and the involvement of PIF4 in the regulation of auxin biosynthesis by cytokinin[J]. Scientific Reports, 2016(6):36-66.
[22]Woodward A W, Bartel B. Auxin: regulation, action, and interaction[J]. Ann Bot, 2005, 95: 707-735.
[23]Vanneste S, Friml J. Auxin: a trigger for change in plant development[J]. Cell, 2009,136: 1005-1016.