中国水仙可溶性淀粉合成酶基因NtSSS的克隆和表达分析
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摘要
以中国水仙‘金盏银台’的花芽为实验材料,采用RT-PCR技术克隆获得可溶性淀粉合成酶基因NtSSS,开放阅读框1 860 bp,编码620个氨基酸。生物信息学分析表明NtSSS编码的氨基酸序列中包含有2个保守基序-淀粉合成酶催化域(Glyco_transf_5)和活性糖基转移域(Glycos_transf_1),其氨基酸序列与凤梨、小兰屿蝴蝶兰和拟兰等单子叶植物较为相似,而与芦笋SSS蛋白的亲缘关系最近。通过荧光定量PCR技术分析了NtSSS在中国水仙不同组织器官和花芽分化不同发育阶段的表达模式,结果表明NtSSS在花蕾中的表达量显著高于茎、叶、根和成熟花中的表达量;在花芽分化发育期NtSSS基因的表达量较低,而在花器官发育期表达量较高,显示出NtSSS基因与中国水仙花器官的发育有关。
I n this study, soluble starch synthase gene NtSSS was cloned by RT-PCR using the flower buds of Narcissus tazetta var. chinensis 'Asclepias curassavica cv. Flaviflora' as the experimental material. The NtSSS gene had an open reading frame of 1 860 bp and encoded 620 amino acids. Bioinformatics analysis showed that the amino acid sequence encoded by NtSSS contained two conservative amino acid sequence-catalytic domain of starch synthase catalytic domain(Glyco_transf_5) with active glycogen transfer domain(Glycos_transf_1). The amino acid sequence of NtSSS was similar with the SSS gene of monocotyledon plants such as Ananas comosus,Phalaenopsis equestris, Apostasia odorata, etc., but the daffodils and asparagus are most closely in genetic relationship. The expression pattern of NtSSS gene in different developmental stages of different organs and flower buds was analyzed by fluorescence quantitative PCR. The results showed that the expression of NtSSS in flower buds was significantly higher than that in stem, leaf, root and mature flower. The expression of NtSSS gene in flower bud differentiation development period was low, but the expression was higher in floral organ development period, which indicated that NtSSS gene might be related to the development of Narcissus tazetta floral organ.
I n this study, soluble starch synthase gene NtSSS was cloned by RT-PCR using the flower buds of Narcissus tazetta var. chinensis 'Asclepias curassavica cv. Flaviflora' as the experimental material. The NtSSS gene had an open reading frame of 1 860 bp and encoded 620 amino acids. Bioinformatics analysis showed that the amino acid sequence encoded by NtSSS contained two conservative amino acid sequence-catalytic domain of starch synthase catalytic domain(Glyco_transf_5) with active glycogen transfer domain(Glycos_transf_1). The amino acid sequence of NtSSS was similar with the SSS gene of monocotyledon plants such as Ananas comosus,Phalaenopsis equestris, Apostasia odorata, etc., but the daffodils and asparagus are most closely in genetic relationship. The expression pattern of NtSSS gene in different developmental stages of different organs and flower buds was analyzed by fluorescence quantitative PCR. The results showed that the expression of NtSSS in flower buds was significantly higher than that in stem, leaf, root and mature flower. The expression of NtSSS gene in flower bud differentiation development period was low, but the expression was higher in floral organ development period, which indicated that NtSSS gene might be related to the development of Narcissus tazetta floral organ.
引文
Chen J.,2012,Study on flower bud differentiation and embryology of Narcissus tazetta var.chinensis,Thesis for M.S.,School of Hortic ulture,Jilin University,Supervisor:Guo T.J.,pp.10-11(陈静,2012,中国水仙花芽分化及胚胎学研究,硕士学位论文,吉林大学园艺学院,导师:郭太君,pp.10-11)
Chen L.J.,Tian H.Q.,and Wu J.,2003,The study on RAPD fingerprints of Narcissus in China and Europe,Redai Yaredai Zuowu Xuebao(Journal of Tropical and Subtropical Botany),11(2):177-180(陈林姣,田惠桥,武剑,2003,中国水仙与欧洲水仙品种RAPD指纹的研究,热带亚热带植物学报,11(2):177-180)
Commuri P.D.,and Keeling P.L.,2001,Chain-length specificities of maize starch synthase I enzyme:studies of glucan affinity and catalytic properties,Plant J.,25(5):475-486
Dong Q.,2015,Identification and characterization of regulatory genes related to starch synthesis in maize(Zea may L.),Dissertation for Ph.D.,School of Life Sciences,Anhui Agricul tural University,Supervisor:Cheng B.J.,pp.1-2(董庆,2015,玉米淀粉合成调控基因的挖掘及功能分析,博士学位论文,安徽农业大学生命科学学院,导师:程备久,pp.1-2)
Gao Z.Y.,Huang D.N.,and Qian Q.,2004,Advances in research on biosynthesis of plant amylopectin,Zhiwu Shengli Yu Fenzi Shengwuxue Xuebao(Journal of Plant Physiology and Molecular Biology),30(5):489-495(高振宇,黄大年,钱前,2004,植物支链淀粉生物合成研究进展,植物生理与分子生物学学报,30(5):489-495)
Horikoshi H.M.,Sekozawa Y.,and Sugaya S.,2017,Inhibition of carbohydrate metabolism by thermal fluctuations during endodormancy lead to negative impacts on bud burst and incidence of floral necrosis in'Housui'Japanese pear flower buds,Sci.Hortic.,224:324-331
James M.G.,Denyer K.,and Myers A.M.,2003,Starch synthesis in the cereal endosperm,Curr.Opin.Plant Biol.,6(3):215-222
Jiang L.,2007,The studies of allelic variation and function of the OsSSS I gene encoding soluble starch synthase I in rice(Oryza sativa L.),Thesis for M.S.,Yangzhou University,Supervisor:Liu Q.Q.,pp.8-12(姜丽,2007,水稻淀粉合成酶OsSSS I基因的等位变异与功能研究,硕士学位论文,扬州大学,导师:刘巧泉,pp.8-12)
Kossmann J.,and Lloyd J.,2000,Understanding and influencing starch biochemistry,Crit.Rev.Biochem.Mol.Biol.,35(3):141-196
Meng X.Q.,Hou Z.X.,and Zhang L.,2013,Progress on molecular and genetic mechanisms of floral control in higher plants,Shengwu Jishu Jinzhan(Current Biotechnology),3(1):1-6(孟晓庆,侯智霞,张兰,2013,高等植物成花转变分子调控机制研究进展,生物技术进展,3(1):1-6)
Myers A.M.,Morell M.K.,James M.G.,and Ball S.G.,2000,Recent progress toward understanding biosynthesis of the amylopectin crystal,Plant Physiol.,122(4):989-997
Smith A.M.,Denyer K.,and Martin C.,1997,The synthesis of the starch granule,Annu.Rev.Plant Physiol.Plant Mol.Biol.,48:67-87
Tan C.X.,Feng C.N.,Chen J.,Guo J.,Guo W.S.,Zhu X.K.,Li C.Y.,and Peng Y.X.,2008,Progress on key enzymes involved in crop starch synthesis and their gene expression,Mailei Zuowu Xuebao(Journal of Triticeae Crops),28(5):912-919(谭彩霞,封超年,陈静,郭静,郭文善,朱新开,李春燕,彭永欣,2008,作物淀粉合成关键酶及其基因表达的研究进展,麦类作物学报,25(5):912-919)
Wang L.X.,Li B.,Yang S.W.,Cao J.L.,Liu X.H.,and Zhang K.,2014,Optimization of capillary electrophoresis for the determination of amylose/amylopectin content in Chinese yam starch,Xiandai Shipin Keji(Modern Food Science and Technology),30(12):282-286(王丽霞,李彬,杨事维,曹金丽,刘相花,张凯,2014,优化毛细管电泳法测定山药淀粉中直链/支链淀粉,现代食品科技,30(12):282-286)
Wu J.H.,Wu S.H.,and Yang C.,2011,Cloning and analysis of RGAs genes coding serine/threonine protein kinase-like proteins from Narcissus tazetta var.chinensis Roem,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),32(5):866-869(吴菁华,吴少华,杨超,2011,中国水仙SKT类抗病基因同源序列的克隆与分析,热带作物学报,32(5):866-869)
Zhang Y.,and Liu Q.L.,2003,Proceedings on molecular mechanism of plant flower development,Zhiwuxue Tongbao(Chinese Bulletin of Botany),20(5):589-601(张云,刘青林,2003,植物花发育的分子机理研究进展,植物学通报,20(5):589-601)
Zhao Q.F.,Chen Y.P.,Gu W.L.,Zhao Q.Y.,Wang H.,Wang H.,Zhu Z.H.,and Song Y.H.,2015,Change of protein and carbohydrate in Vanilla planifolia andrews during flower bud differentiation period,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),36(6):1053-1058(赵秋芳,陈娅萍,顾文亮,赵青云,王辉,王华,朱自慧,宋应辉,2015,香草兰花芽分化期蛋白质及碳水化合物变化研究,热带作物学报,36(6):1053-1058)
Chen L.J.,Tian H.Q.,and Wu J.,2003,The study on RAPD fingerprints of Narcissus in China and Europe,Redai Yaredai Zuowu Xuebao(Journal of Tropical and Subtropical Botany),11(2):177-180(陈林姣,田惠桥,武剑,2003,中国水仙与欧洲水仙品种RAPD指纹的研究,热带亚热带植物学报,11(2):177-180)
Commuri P.D.,and Keeling P.L.,2001,Chain-length specificities of maize starch synthase I enzyme:studies of glucan affinity and catalytic properties,Plant J.,25(5):475-486
Dong Q.,2015,Identification and characterization of regulatory genes related to starch synthesis in maize(Zea may L.),Dissertation for Ph.D.,School of Life Sciences,Anhui Agricul tural University,Supervisor:Cheng B.J.,pp.1-2(董庆,2015,玉米淀粉合成调控基因的挖掘及功能分析,博士学位论文,安徽农业大学生命科学学院,导师:程备久,pp.1-2)
Gao Z.Y.,Huang D.N.,and Qian Q.,2004,Advances in research on biosynthesis of plant amylopectin,Zhiwu Shengli Yu Fenzi Shengwuxue Xuebao(Journal of Plant Physiology and Molecular Biology),30(5):489-495(高振宇,黄大年,钱前,2004,植物支链淀粉生物合成研究进展,植物生理与分子生物学学报,30(5):489-495)
Horikoshi H.M.,Sekozawa Y.,and Sugaya S.,2017,Inhibition of carbohydrate metabolism by thermal fluctuations during endodormancy lead to negative impacts on bud burst and incidence of floral necrosis in'Housui'Japanese pear flower buds,Sci.Hortic.,224:324-331
James M.G.,Denyer K.,and Myers A.M.,2003,Starch synthesis in the cereal endosperm,Curr.Opin.Plant Biol.,6(3):215-222
Jiang L.,2007,The studies of allelic variation and function of the OsSSS I gene encoding soluble starch synthase I in rice(Oryza sativa L.),Thesis for M.S.,Yangzhou University,Supervisor:Liu Q.Q.,pp.8-12(姜丽,2007,水稻淀粉合成酶OsSSS I基因的等位变异与功能研究,硕士学位论文,扬州大学,导师:刘巧泉,pp.8-12)
Kossmann J.,and Lloyd J.,2000,Understanding and influencing starch biochemistry,Crit.Rev.Biochem.Mol.Biol.,35(3):141-196
Meng X.Q.,Hou Z.X.,and Zhang L.,2013,Progress on molecular and genetic mechanisms of floral control in higher plants,Shengwu Jishu Jinzhan(Current Biotechnology),3(1):1-6(孟晓庆,侯智霞,张兰,2013,高等植物成花转变分子调控机制研究进展,生物技术进展,3(1):1-6)
Myers A.M.,Morell M.K.,James M.G.,and Ball S.G.,2000,Recent progress toward understanding biosynthesis of the amylopectin crystal,Plant Physiol.,122(4):989-997
Smith A.M.,Denyer K.,and Martin C.,1997,The synthesis of the starch granule,Annu.Rev.Plant Physiol.Plant Mol.Biol.,48:67-87
Tan C.X.,Feng C.N.,Chen J.,Guo J.,Guo W.S.,Zhu X.K.,Li C.Y.,and Peng Y.X.,2008,Progress on key enzymes involved in crop starch synthesis and their gene expression,Mailei Zuowu Xuebao(Journal of Triticeae Crops),28(5):912-919(谭彩霞,封超年,陈静,郭静,郭文善,朱新开,李春燕,彭永欣,2008,作物淀粉合成关键酶及其基因表达的研究进展,麦类作物学报,25(5):912-919)
Wang L.X.,Li B.,Yang S.W.,Cao J.L.,Liu X.H.,and Zhang K.,2014,Optimization of capillary electrophoresis for the determination of amylose/amylopectin content in Chinese yam starch,Xiandai Shipin Keji(Modern Food Science and Technology),30(12):282-286(王丽霞,李彬,杨事维,曹金丽,刘相花,张凯,2014,优化毛细管电泳法测定山药淀粉中直链/支链淀粉,现代食品科技,30(12):282-286)
Wu J.H.,Wu S.H.,and Yang C.,2011,Cloning and analysis of RGAs genes coding serine/threonine protein kinase-like proteins from Narcissus tazetta var.chinensis Roem,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),32(5):866-869(吴菁华,吴少华,杨超,2011,中国水仙SKT类抗病基因同源序列的克隆与分析,热带作物学报,32(5):866-869)
Zhang Y.,and Liu Q.L.,2003,Proceedings on molecular mechanism of plant flower development,Zhiwuxue Tongbao(Chinese Bulletin of Botany),20(5):589-601(张云,刘青林,2003,植物花发育的分子机理研究进展,植物学通报,20(5):589-601)
Zhao Q.F.,Chen Y.P.,Gu W.L.,Zhao Q.Y.,Wang H.,Wang H.,Zhu Z.H.,and Song Y.H.,2015,Change of protein and carbohydrate in Vanilla planifolia andrews during flower bud differentiation period,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),36(6):1053-1058(赵秋芳,陈娅萍,顾文亮,赵青云,王辉,王华,朱自慧,宋应辉,2015,香草兰花芽分化期蛋白质及碳水化合物变化研究,热带作物学报,36(6):1053-1058)