STM和CUC2基因对不同抗寒性白菜型冬油菜生长点的调控研究
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摘要
为研究抗寒性强的白菜型冬油菜(Brassica rapa)调控生长点(shoot apical meristem,SAM)凹陷生长的SHOOT MERISTEMLESS(STM)和CUP-SHAPED COTYLEDON 2(CUC2)基因,采用RT-PCR技术克隆两基因并进行了表达分析。STM基因CDS序列开放阅读框长度为1 554bp,编码517个氨基酸,该基因在进化上高度保守,其序列属于PLN03226超家族。CUC2基因开放阅读框长度为1 104bp,编码367个氨基酸,该基因保守序列属于NAM超家族,是一个主要由不规则卷曲与延伸链组成的亲水性蛋白。荧光定量分析表明,相同温度处理下,STM基因在抗寒性较强、生长点凹陷的白菜型冬油菜陇油6号和陇油7号中相对表达量均低于抗寒性较弱、生长点凸起的天油4号和Lenox,而CUC2基因相对表达量的变化趋势与STM基因相反;低温(0℃、4℃和-4℃)处理72h后,白菜型冬油菜生长点中STM、CUC2基因均上调表达,表明STM和CUC2基因可能参与抗寒应答及白菜型冬油菜生长点对低温胁迫的响应。
To understand the molecular regulation of SHOOT MERISTEMLESS(STM) and CUP-SHAPED COTYLEDON 2(CUC2) genes on shoot apical meristem growth, both genes were cloned from winter Brassica rapa by RT-PCR. CDS sequence of STM gene was obtained to be of 1 554 bp in length, encoding 517 amino acids, which was highly evolutionally conserved and belonged to PLN03226 superfamily. CUC2 open reading frame was 1 104 bp long, encoding 367 amino acids, belonging to NAM superfamily. CUC2 was predicted to be a hydrophilic protein mainly composed of irregular curly and extended chains. Fluorescence quantitative analysis showed that under the same temperature, the relative expression of STM gene in winter B.rapa(cvs Longyou 6 and Longyou 7 with strong cold resistances, which had recessed growth cones) were lower than that with raised cone in Tianyou 4 and Lenox. But the relative expression of CUC2 gene was the opposite. After 72 h of cold(0℃, 4℃ and-4℃) treatment, STM and CUC2 genes in the growth cones were up-regulated, indicating that both genes were involved in cold response in the growth cone to low temperature.
To understand the molecular regulation of SHOOT MERISTEMLESS(STM) and CUP-SHAPED COTYLEDON 2(CUC2) genes on shoot apical meristem growth, both genes were cloned from winter Brassica rapa by RT-PCR. CDS sequence of STM gene was obtained to be of 1 554 bp in length, encoding 517 amino acids, which was highly evolutionally conserved and belonged to PLN03226 superfamily. CUC2 open reading frame was 1 104 bp long, encoding 367 amino acids, belonging to NAM superfamily. CUC2 was predicted to be a hydrophilic protein mainly composed of irregular curly and extended chains. Fluorescence quantitative analysis showed that under the same temperature, the relative expression of STM gene in winter B.rapa(cvs Longyou 6 and Longyou 7 with strong cold resistances, which had recessed growth cones) were lower than that with raised cone in Tianyou 4 and Lenox. But the relative expression of CUC2 gene was the opposite. After 72 h of cold(0℃, 4℃ and-4℃) treatment, STM and CUC2 genes in the growth cones were up-regulated, indicating that both genes were involved in cold response in the growth cone to low temperature.
引文
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[24] 乌凤章,王柏臣,刘桂丰,等.低温胁迫对白桦幼苗生长和生理的影响[J].东北林业大学学报,2008,36(9):8-10.
[25] 孙万仓,武军艳,曾军,等.8 个白菜型冬油菜品种抗寒性的初步评价[J].湖南农业大学学报(自然科学版),2007 (S1).
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[27] 武军艳,方彦,张朋飞,等.北方旱寒区冬油菜根系抗寒指标分析[J].干旱地区农业研究,2014,32(6):250-255.
[28] Long J,Barton M.K.Initiation of axillary and floral meristems in Arabidopsis[J].Dev Biol,2002,218:341-353.
[29] Barton M K.Cell type specification and self renewal in the vegetative shoot apical meristem[J].Curr Opin Plant Biol,1998,1(1):37-42.
[30] Lenhard M,Laux T.Shoot meristem formation and maintenance[J].Curr Opin Plant Biol,1999,2(1):44-50.
[31] Veit B.Hormone mediated regulation of the shoot meristem[J].Plant Mol Biol,2009,69:397-408.
[32] Jackson D,Veit B,Hake S.Expression of maize KNOTTED1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot[J].Development,1994,120:405-413.
[33] Long J A.The development of apical embryonic pattern in Arabidopsis[J].Development,1998,125:3 027-3 035.
[34] Aida M,Ishida T,Fukaki H,et al.Genes involved in organ separation in Arabidopsis:an analysis of the cup-shaped cotyledon mutant[J].The Plant Cell,1997,9(6):841-857.
[2] 马骊,孙万仓,刘自刚,等.白菜型与甘蓝型冬油菜抗寒机理差异的研究[J].华北农学报,2016,31(1):147-154.
[3] 刘海卿,孙万仓,刘自刚,等.北方寒旱区白菜型冬油菜抗寒性与抗旱性评价及其关系[J].中国农业科学,2015,48(18):3 743-3 756.
[4] 崔玉超,陈亮.拟南芥茎尖干细胞调控机制的研究进展[J].厦门大学学报:自然科学版,2016,55(6):781-792.
[5] Guo M,Thomas J,Collins G.et al.Direct repression of KNOX loci by the ASYMMETRIC LEAVES1 complex of Arabidopsis[J].The Plant Cell,2008,20:48-58.
[6] Gallois J L,Woodward C,Reddy G V,et al.Combined SHOOT MERISTEMLESS and WUSCHEL trigger ectopic organogenesis in Arbidopsis[J].Development,2002,129:3 207-3 217.
[7] Byrne M E,Simorowski J,Martienssen R A.ASYMMETRIC LEAVES1 reveals knox gene redundancy in Arabidopsis[J].Development,2002,129:1 957-1 965.
[8] Balkunde R,Kitagawa M,Xu X M,et al.SHOOT MERISTEMLESS trafficking controls axillary meristem formation,meristem size and organ boundaries in Arabidopsis[J].Plant J Cell Mol Biol,2017,90:435-446.
[9] 王珊珊.拟南芥TIR3基因调控茎端分生组织和胚胎发育的机理研究[D].泰安:山东农业大学,2018.
[10] Lenhard M,Jürgens G,Laux T.The WUSCHEL and SHOOTMERISTEMLESS genes fulfil complementary roles in Arabidopsis shoot meristem regulation[J].Development,2002,129:3 195-3 206.
[11] Brand U,Grünewald M,Hobe M.et al.Regulation of CLV3 expression by two homeobox genes in Arabidopsis[J].Plant Physiol,2002,129:565-575.
[12] Kwon C S,Hibara K,Pfluger J,et al.A role for chromatin remodeling in regulation of CUC gene expression in the Arabidopsis cotyledon boundary[J].Development,2006,133:3 223-3 230.
[13] Bowman J L,Eshed Y.Formation and maintenance of the shoot apical meristem[J].Trends Plant Sci,2000,5(3):110-115.
[14] Aida M,Ishida T,Tasaka M.Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis:interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes[J].Development,1999,126(8):1 563-1 570.
[15] Kamiuchi Y,Yamamoto K,Furutani M,et al.The CUC1 and CUC2 genes promote carpet margin meristem formation during Arabidopsis gynoecium development[J].Front Plant Sci,2014:5(165):1-9.
[16] Nikovics K ,Blein T ,Peaucelle A ,et al.The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis[J].Plant Cell,2006,18(11):2 929-2 945.
[17] Spinelli S V,Martin A P,Viola I L,et al.A mechanistic link between STM and CUC1 during Arabidopsis development[J].Plant Physiol,2011,156:1 894-1 904.
[18] 马骊,袁金海,孙万仓,等.白菜型冬油菜类甜蛋白的筛选、克隆及其在低温胁迫下的表达[J].作物学报,2017,43(4):620-628.
[19] 张书,乐愉,武斐.陆地棉GRF基因家族的鉴定和生物信息学分析[J].分子植物育种,2019,26(2):1-10.
[20] 鲁丹丹,李保全,安素妨,等.甘蓝型油菜BnDMC1.A01基因的分离及序列分析[J].中国油料作物学报,2018,40(6):745-754.
[21] 马骊,孙万仓,袁金海,等.白菜型冬油菜β-1,3-葡聚糖酶基因在低温胁迫下的表达[J].植物学报,2017,52(05):568-578.
[22] 王学芳.我国西北白菜型和甘蓝型冬油菜品种抗寒性研究[D].兰州:甘肃农业大学,2009.
[23] 宋绪忠.茶树无性系苗期抗寒特性研究[D].泰安:山东农业大学,2002.
[24] 乌凤章,王柏臣,刘桂丰,等.低温胁迫对白桦幼苗生长和生理的影响[J].东北林业大学学报,2008,36(9):8-10.
[25] 孙万仓,武军艳,曾军,等.8 个白菜型冬油菜品种抗寒性的初步评价[J].湖南农业大学学报(自然科学版),2007 (S1).
[26] 杨宁宁,孙万仓,刘自刚,等.北方冬油菜抗寒性的形态与生理机制[J].中国农业科学,2013,47(3):452-461.
[27] 武军艳,方彦,张朋飞,等.北方旱寒区冬油菜根系抗寒指标分析[J].干旱地区农业研究,2014,32(6):250-255.
[28] Long J,Barton M.K.Initiation of axillary and floral meristems in Arabidopsis[J].Dev Biol,2002,218:341-353.
[29] Barton M K.Cell type specification and self renewal in the vegetative shoot apical meristem[J].Curr Opin Plant Biol,1998,1(1):37-42.
[30] Lenhard M,Laux T.Shoot meristem formation and maintenance[J].Curr Opin Plant Biol,1999,2(1):44-50.
[31] Veit B.Hormone mediated regulation of the shoot meristem[J].Plant Mol Biol,2009,69:397-408.
[32] Jackson D,Veit B,Hake S.Expression of maize KNOTTED1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot[J].Development,1994,120:405-413.
[33] Long J A.The development of apical embryonic pattern in Arabidopsis[J].Development,1998,125:3 027-3 035.
[34] Aida M,Ishida T,Fukaki H,et al.Genes involved in organ separation in Arabidopsis:an analysis of the cup-shaped cotyledon mutant[J].The Plant Cell,1997,9(6):841-857.