作物杂志, 2018, 34(6): 116-123 doi: 10.16035/j.issn.1001-7283.2018.06.018

生理生化·植物营养·栽培耕作

外源过氧化氢对烟草花芽分化的影响初探

王宁, 张静, 黄进勇, 史团省, 杜建, 岳彩鹏

郑州大学生命科学学院,450001,河南郑州

Effects of Exogenous Hydrogen Peroxide on Floral Bud Differentiation in Tobacco

Wang Ning, Zhang Jing, Huang Jinyong, Shi Tuansheng, Du Jian, Yue Caipeng

School of Life Sciences, Zhengzhou University, Zhengzhou 450001, Henan, China

通讯作者: 岳彩鹏,副教授,主要从事植物生理与分子生物学研究

收稿日期: 2018-09-30   网络出版日期: 2018-12-15

基金资助: 河南省基础与前沿技术研究计划项目.  162300410176

Received: 2018-09-30   Online: 2018-12-15

作者简介 About authors

王宁,硕士,主要从事植物生理研究;张静为共同第一作者,硕士,主要从事植物生理研究 。

摘要

通过盆栽试验,对外源H2O2处理的烟草活性氧代谢及烟草花芽分化情况进行了初步研究。结果表明:(1)外源H2O2处理显著影响了烟草体内活性氧代谢,H2O2和O2-含量迅速增加;保护酶POD和CAT活性迅速被激活,SOD的激活滞后于POD和CAT。喷施H2O2处理的中后期H2O2的含量下降。(2)外源H2O2胁迫改变了开花基因的表达、现蕾时烟草叶片数以及花芽的发育进程。5和15d的外源H2O2处理明显抑制了烟草FLC基因的表达,促进了LFY基因的表达,促使烟草花发育提前。试验结果表明烟草体内活性氧平衡状态的变化影响烟草的花发育进程。

关键词: 烟草 ; 外源过氧化氢 ; 活性氧 ; 花芽分化

Abstract

The active oxygen metabolism and flower bud differentiation in tobacco were studied by spraying exogenous H2O2 via pot experiments. Results showed that: (1) The active oxygen metabolism was significantly changed in tobacco by spraying H2O2. After spraying H2O2, the content of H2O2 and O2_ was rapidly increased in tobacco, the activities of POD and CAT were rapidly activated followed by the activation of SOD. In the middle and latter period of spraying, the content of O2_ was high, while the content of H2O2 was significantly decreased. (2) The expressions of the floral development genes were affected, the number of leaves in squaring stage was altered and the process of flower bud development was modified by spraying of H2O2. The expression of FLC gene was significantly inhibited while the expression of LFY gene was significantly promoted in tobaccos which were sprayed H2O2 at 5 and 15 consecutive days. Floral bud differentiation of tobaccos progressed earlier under 5 to 25 consecutive days spray of H2O2. The flowering transition was affected by the change of active oxygen equilibrium in tobacco.

Keywords: Tobacco ; Exogenous hydrogen peroxide ; Reactive oxygen species ; Floral bud differentiation

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本文引用格式

王宁, 张静, 黄进勇, 史团省, 杜建, 岳彩鹏. 外源过氧化氢对烟草花芽分化的影响初探[J]. 作物杂志, 2018, 34(6): 116-123 doi:10.16035/j.issn.1001-7283.2018.06.018

Wang Ning, Zhang Jing, Huang Jinyong, Shi Tuansheng, Du Jian, Yue Caipeng. Effects of Exogenous Hydrogen Peroxide on Floral Bud Differentiation in Tobacco[J]. Crops, 2018, 34(6): 116-123 doi:10.16035/j.issn.1001-7283.2018.06.018

活性氧(ROS)是植物有氧代谢的副产物,主要包括过氧化氢(H2O2)、超氧阴离子(O2-)、羟自由基(OH·)和单线态氧(1O2[1,2],当植物遭遇如干旱、盐害、冷害、热击、紫外线辐射、臭氧污染、病原体入侵[3]等胁迫时,均会导致呼吸链、光合电子传递链以及细胞质膜上的NADPH氧化酶[4,5,6,7]产生过量ROS,从而伤害膜系统,导致蛋白质、DNA及脂类氧化伤害,最终导致细胞损伤[8,9]

近些年研究发现,活性氧、一氧化氮[10,11,12]、一氧化碳[13]等均为信息分子家族的新成员,通过改变细胞的氧化还原状态、蛋白激酶活性、蛋白磷酸酶活性、离子通道的活性[14]或转录因子的氧化还原状态[15],参与多种信号通路的调控,响应逆境生理过程。H2O2是一种相对稳定的活性氧(半衰期为1ms),在生理pH下不带电荷,分子很小,可以通过质膜自由扩散[16],由于其氧化还原性,它能够很快与植物体内氧化还原信号途径的成分相互作用[17]。作为重要的信号传递物质,H2O2能引起蚕豆保卫细胞的气孔关闭[18],诱导细胞壁的生长与重建[19],促进根和根毛生长[20,21,22],促进细胞伸长和植株生长[23,24]以及促进糊粉层细胞程序性死亡等[25,26],但是关于H2O2对植物花发育的调控研究报道较少。

FLOWERING LOCUS C(FLC)是调节开花时间的开花抑制基因[27,28]。LEAFY(LFY)是重要的花分生组织决定基因,调控植物的成花转变[29,30]

试验探讨外源H2O2胁迫下,烟草体内ROS代谢响应过程及花芽分化情况,同时从分子水平上分析开花基因FLC、LFY转录水平的变化,初步探讨ROS作为信号分子对烟草花发育的调控作用。

1 材料与方法

1.1 材料

以烟草K326作为供试材料,种子由青岛中烟种子公司提供。

1.2 试验方法

试验于2015-2016年在郑州大学生命科学学院农业植物资源研究与利用实验室进行。试验采用格盘漂浮法进行育苗,将烟草K326种子均匀播种于湿润的基质上,根据生长情况定期浇灌Hoagland营养液,于室温25℃条件下生长。

待烟苗长到第10片真叶时,选取长势均匀一致的烟株挂牌标记叶位,随后移栽,将烟株分为处理组和对照组,处理组6个处理,标记为T1、T2、T3、T4、T5、T6,分别喷施H2O2溶液5、10、15、20、25、30d,于每天上午8:00在烟草叶片正反面均匀喷施H2O2溶液(10mmol/L),以溶液不成股流下为宜;对照组(CK)同时喷施等量的清水;每个处理设置3个重复。根据预试验结果和其他相关研究[31,32],H2O2浓度确定为10mmol/L。

每个处理于喷施5、10、15、20、25、30d取新鲜叶片,测定保护酶活性、活性氧含量等生理指标;为了探讨烟草对外源H2O2的快速响应,分别于处理1、2、4、8、12、24h取样,记为H1、H2、H3、H4、H5、H6,测定各生理指标;各组处理结束后,正常条件培养,每天观察记录烟株生长状况,每隔5d观察记录烟草花芽分化情况,适时选取茎尖固定保存,统计烟草现蕾时叶片数;选取处理T1、T3和CK,分别于喷施5、10、15、20、25d时取茎尖,对开花抑制基因FLC、花分生组织决定基因LFY的表达情况进行分析。

1.3 测定指标及方法

生理指标测定:采用TBA法测定丙二醛(MDA)的含量[33],采用氮蓝四唑法测定超氧化物歧化酶(SOD)的活性[34],采用愈创木酚法测定过氧化物酶(POD)的活性[34],采用紫外吸收法测定过氧化氢酶(CAT)的活性[34],采用羟胺氧化法测定氧自由基(O2-)的含量[35],采用比色法测定H2O2的含量[36]

形态学指标鉴定:花芽解剖结构的观察,采用体式显微镜(50×Nikon SMZ800)进行观察。FAA固定液的配比是:38%甲醛5.0mL,冰乙酸5.0mL,75%乙醇90.0mL[37]。根据烟株上挂牌标记的叶位观察并记录烟草现蕾时的叶片数,每个处理组以每组各株现蕾时叶片数的平均值记。

开花基因表达量的测定:从NCBI网站GenBank查找已登记基因LFY(GenBank:JQ686928.1)和β-actin(GenBank:NO.U60495)的核酸序列,通过软件DNAman设计引物,FLC引物的设计参照李元元[38]。LFY基因引物为F:TAAGCCAAAAATGCGACACT,R:GTTCAGAATGGCAAAGCTGG;FLC基因引物为F:CTCAAGAAAATAGCAGCCTTCC,R:TCTCCTTATTGCTCCTCACACA;内参β-actin基因引物为F:ATGCCTATGTGGGTGACGAAG,R:TCTGTTGGCCTTAGGGTTGAG,送生工生物工程(上海)股份有限公司合成。郑州贝贝生物科技有限公司Total RNA提取试剂盒提取总RNA,北京爱普拜生物技术有限公司的Hiscript cDNA试剂盒反转录,FastStart Universal SYBR Green Master(Roche)试剂盒实时荧光定量PCR,Roche Light Cycler®480 II荧光定量PCR。20μL反应体系为:HiTaq SYBR Green Mastermix(2x)10μL,Forward Primer(10mM)0.4μL,Reverse Primer(10mM)0.4μL,cDNA 1μL,ddH2O 8.2μL。反应程序为:95℃预变性2min;95℃变性15s,60℃退火20s,72℃延伸20s,40个循环。采用2-△△CT法分析结果[39]

1.4 数据处理

采用Excel 2016进行数据处理,SPSS 21.0软件进行数据统计分析,OriginPro 8.5进行作图。

2 结果与分析

2.1 外源H2O2对烟草ROS含量的影响

图1显示,外源H2O2处理组烟草体内O2-含量升高,4h后显著高于对照,处理12h时,O2-含量达到最高水平;处理1、2、4、8、12、24h分别对应9:00、10:00、12:00、16:00、20:00和第二天早上8:00,对照组烟草体内O2-含量的波动显示,烟草体内O2-含量呈随光照的改变而改变的昼夜节律,中午12:00时烟草体内O2-含量最高。图2表明,随着外源H2O2处理时间的延长,20d前烟草体内O2-含量维持在较高水平,极显著高于对照,25d后O2-含量逐渐降低,和对照组差异不显著。

图1

图1   外源H2O2处理烟草O2-含量的快速响应变化

*:0.05水平差异显著;**:0.01水平差异极显著;下同

Fig.1   Rapidly responsive changes of O2- content in tobacco treated with exogenous H2O2

*, Significant difference at 0.05 level; **, Extremely significant difference at 0.01 level; the same below


图2

图2   长时间外源H2O2处理烟草O2-含量的变化

Fig.2   Changes of O2- content in tobacco treated with exogenous H2O2 for a long time


图3表明,外源H2O2处理烟草体内H2O2含量快速反应,1h时迅速升高,极显著高于对照组;随后烟草启动保护机制,H2O2得到清除,喷施2h后,处理组H2O2含量降低,但都显著高于对照组。对照组烟草体内H2O2含量的波动同样显示烟草体内H2O2含量1d内的节律变化,中午12:00时烟草体内H2O2含量最高。

图3

图3   外源H2O2处理烟草H2O2含量的快速响应变化

Fig.3   Rapidly responsive changes of H2O2 content in tobacco treated with exogenous H2O2


长时间H2O2胁迫结果见图4,10d前处理组H2O2含量显著高于对照组,随后和对照组差异不显著。结果表明,外源H2O2处理使烟草体内H2O2含量和O2-含量迅速升高,但二者的变化趋势有所不同,H2O2含量变化更为快速,O2-含量升高略晚,高含量O2-持续的时间较长,因此推测外源H2O2通过改变烟草体内活性氧水平及其平衡状态,进而影响烟草的生长发育进程。

图4

图4   长时间外源H2O2处理烟草H2O2含量的变化

Fig.4   Changes of H2O2 content in tobacco treated with exogenous H2O2 for a long time


2.2 外源H2O2对烟草保护酶系的影响

图5表明,外源H2O2处理1h后烟草细胞POD的活性迅速升高,处理组POD活性极显著高于对照组;处理4h时POD活性达到最大,说明烟草细胞POD能够快速响应外源H2O2的诱导,迅速被激活清除体内H2O2,原因可能是H2O2是其直接作用底物,能够快速启动应激机制。对照组烟草POD活性的波动显示POD活性1d内的节律变化,随着光强增强,中午12:00时活性有所下降,次日上午8:00活性最低。

图5

图5   外源H2O2处理POD活性的快速响应变化

Fig.5   Rapid response changes of POD activity in tobacco treated with exogenous H2O2


图6表明,处理5~15d过程中处理组POD活性逐渐升高,并显著或极显著高于对照组,随着外源H2O2处理时间的延长,20d烟草POD活性下降,但仍极显著高于对照。25和30d处理组POD活性与对照组差异不显著。

图6

图6   长时间外源H2O2处理烟草POD活性的变化

Fig.6   Changes of POD activity in tobacco treated with exogenous H2O2 for a long time


图7显示,烟草细胞CAT对外源H2O2的胁迫诱导比POD响应略慢,喷施后4h H2O2处理组CAT活性开始显著高于对照,喷施后8h CAT活性达到最大,随后活性有所下降,24h时处理组与对照组差别不显著。对照组烟草CAT活性的波动显示1d内CAT活性的节律变化,随着光强增强,中午12:00后活性有所下降,下午4:00活性最低。长时间H2O2处理显示(图8),处理组CAT活性一直维持在较高水平,显著高于对照组,10d时CAT活性最高,之后呈波动性下降。说明在长期H2O2胁迫下,CAT对H2O2的清除发挥着主要的作用。

图7

图7   外源H2O2处理烟草CAT活性的快速响应变化

Fig.7   Rapidly responsive changes of CAT activity in tobacco treated with exogenous H2O2


图8

图8   长时间外源H2O2处理烟草体内CAT活性的变化

Fig.8   Changes of CAT activity in tobacco treated with exogenous H2O2 for a long time


图9显示,外源H2O2处理,烟草SOD响应较慢,处理8h之内处理组与对照组没有显著差异;处理12h后处理组SOD活性极显著高于对照组。对照组烟草SOD活性呈现先升高后下降再升高的节律性变化,随着光强增强,中午12:00活性下降,下午4:00活性最低。长时间外源H2O2处理(图10),处理5~15d烟草SOD活性极显著高于对照组,20d后与对照组差异不显著。可见外源H2O2胁迫可以激活烟草细胞SOD活性,但略滞后于POD和CAT,随着生育期推移和外源活性氧作用时间延长,SOD活性有所下降,不利于活性氧的清除。

图9

图9   外源H2O2处理烟草SOD活性的快速响应变化

Fig.9   Rapidly responsive changes of SOD activity in tobacco treated with exogenous H2O2


图10

图10   长时间外源H2O2处理烟草体内SOD活性的变化

Fig.10   Changes of SOD activity in tobacco treated with exogenous H2O2 for a long time


由于POD和CAT主要是清除烟草体内H2O2,外源H2O2胁迫下,保护酶POD和CAT响应早于SOD,其中POD最为迅速,CAT的响应略晚,但CAT的高活性比POD维持的时间较长,说明在清除H2O2过程中CAT和POD协同发挥着关键作用。

2.3 外源H2O2对基因FLC、LFY转录表达的影响

随着烟草生育期的推进,开花抑制基因FLC表达量逐渐下降,外源H2O2胁迫明显影响了FLC的表达。如图11所示,T1组和T3组FLC的表达量低于对照,说明处理组FLC对花发育的抑制作用减小,使得花芽分化提前。

图11

图11   外源H2O2对基因FLC表达量的影响

Fig.11   Effect of exogenous H2O2 on the expression of FLC


外源H2O2对花分生组织决定基因LFY表达量的影响如图12所示,在外源H2O2作用下LFY表达量呈升高趋势,T1组处理后第10天LFY表达量开始增强,20、25d后其表达量明显高于对照组;T3组的LFY表达量在处理第25天明显高于对照。结果表明,一定程度的外源H2O2处理抑制了开花抑制因子FLC的表达,促进了烟草花分生组织决定基因LFY的表达,从而促进烟草成花转变;但H2O2胁迫时间过长,不利于LFY表达,影响花的发育。

图12

图12   外源H2O2对基因LFY表达量的影响

Fig.12   Effect of exogenous H2O2 on the expression of LFY


2.4 外源H2O2对烟草花芽分化的影响

外源H2O2处理45d时不同处理烟草花芽分化情况见图13,花芽分化观察结果的分析参照相关烟草花芽分化的显微观察研究[37,40],对照组烟草茎顶端生长锥处于营养生长阶段的后期,顶端分生组织略突起,周缘着生3个叶原基;处理1d(H6)的烟株5个花萼原基基本形成,花瓣原基开始分化;T1已形成雄蕊原基和雌蕊原基,雄蕊原基内侧开始形成凹陷纵沟,雌蕊柱头清晰可见,中间凹陷二裂;T2、T3都处于花瓣、雄蕊原基分化时期,花瓣雄蕊交互排列,5个雄蕊原基形成,内侧还未凹陷;T4花萼原基分化完成,开始花瓣原基和雄蕊原基分化;T5顶端分生组织中间半球形突起,处于花萼原基分化期,第5个萼片正在形成;T6顶花原基分化初期,侧枝原基正在分化。花芽分化镜检结果显示,H2O2处理组烟草的花芽分化不同程度地早于对照组,其中T1处理组花芽分化进展最早,其次是T2和T3处理组。表明外源H2O2胁迫,促进烟草花发育进程。

图13

图13   外源H2O2条件下花芽分化情况(10×5)

Fig.13   The flower bud differentiation of tobacco under exogenous H2O2 conditions (10×5)


图14显示外源H2O2喷施5、10、15、20、25d的烟草都出现花前叶片数减少的现象,现蕾时叶片数分别比对照组减少了5、6、5、3、2片,其中T1、T2、T3组与对照CK的差异达到极显著、显著水平。可见,一定程度的H2O2胁迫可以促进烟草花芽发育,表明超氧阴离子与H2O2代谢的失衡对促进烟草花发育启动具有调控作用。

图14

图14   外源H2O2处理烟草现蕾时叶片数

Fig.14   The leaf number of tobacco when buds occur under exogenous H2O2


3 结论与讨论

研究结果表明,外源H2O2处理显著改变了烟草体内活性氧代谢,H2O2含量迅速升高,随后O2-含量也开始积累。外源H2O2胁迫1h,烟草保护酶POD活性迅速被激活,在前期清除H2O2过程中发挥主要作用;处理4h后CAT活性被激活,在中后期其活性的增加幅度远远高于POD,在中后期清除H2O2过程中发挥主要作用;SOD对外源H2O2的响应晚于POD和CAT,可能H2O2的积累影响了O2-的代谢所致,长期H2O2处理后期SOD活性逐渐下降,趋于稳定。保护酶SOD、CAT和POD的活性与植物体内的活性氧代谢直接相关,在一定程度上代表植物体清除活性氧自由基的能力,反映植物抗逆性的强弱[41]。卢金[42]研究报道,小麦“宁春”在外源H2O2处理细胞内SOD、APX和GR的活性升高,而小麦“西旱”则是提高CAT、APX和GR活性来清除H2O2,证明了APX与GR在清除活性氧中也具有十分重要的作用。H2O2在植物体内具有双重作用,高浓度H2O2对植物细胞会造成氧化损伤,而低浓度的H2O2预处理能增加植物的非酶抗氧化物质ASA和GSH的含量以及抗氧化酶活性,提高植物抗性[43]

近年来研究报道,ROS调节植物多种发育过程,如细胞的增殖与分化、种子萌发、气孔关闭、根向地性、花粉管的生长和植物衰老等[44]。野燕麦种子糊粉层中ROS含量的增加,以及SOD和CAT活性的降低,可能导致细胞程序性死亡[45]。赵士诚等[46]研究发现镉影响玉米幼苗的活性氧代谢,玉米幼苗经短时间镉处理,其体内活性氧含量增加,SOD和CAT活性及其酶基因的表达都有所增加,表明氧化胁迫调控保护酶基因的表达。H2O2被认为是多种生理生化过程中的重要信号物质,包括胁迫应答和防御反应、信号转导、代谢以及生长发育等[47]。周碧燕课题组进行的荔枝盆栽试验证明,中温组的树木进行ROS处理后促进了开花[48]。岳彩鹏课题组研究发现,低温胁迫引起烟草体内活性氧代谢失衡,烟草现蕾时叶片数减少,烟草花芽分化提前完成[36]

拟南芥中已经鉴定出FLC、FT、SOC1、LFY和SVP等5个开花整合子,编码植物特有的转录因子,在生殖生长过程中扮演着开花时间促进基因和花分生组织决定基因双重角色[49]。FLC是植物中普遍存在的开花抑制基因,在植物营养生长阶段高表达,抑制下游花发育正调控因子的表达,维持植物的营养生长[27,28]。LFY是花发育正调控因子,在成花转变早期表达增强,正调控下游花器官发育基因的表达,促进花发育[29,30]。试验表明喷施外源H2O2使烟草体内活性氧状态失衡,一定程度地促进了烟草花芽分化。喷施H2O2 5~25d的处理,烟草现蕾时叶片数减少,烟草花芽分化提前。喷施5和15d的H2O2处理组开花抑制基因FLC表达明显下调,而LFY表达明显上调,从而促进烟草的花发育。外源H2O2处理对烟草体内活性氧代谢的影响结果显示,烟草体内活性氧在喷施一段时间内含量升高,喷施结束后,在烟草自身清除体系作用下逐渐恢复正常。由此分析一定程度的活性氧变化能够作为一种信号,负调控FLC的表达,促进LFY表达,从而促进了烟草的花芽分化进程;喷施H2O2时间过长,对烟草的氧化损伤较大,不利于开花相关基因的表达。试验结果表明活性氧信号在转录水平上,通过调控开花整合子FLC和LFY的表达参与烟草花芽分化过程,本研究初步表明了H2O2调控植物花发育的生理和分子机理,植物开花的活性氧调控机制和信号通路还需要进一步深入探索。

The authors have declared that no competing interests exist.
作者已声明无竞争性利益关系。

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张静, 李园园, 岳彩鹏 , .

低温胁迫下活性氧代谢与烟草花芽分化的研究

作物杂志, 2015(4):74-80.

DOI:10.16035/j.issn.1001-7283.2015.04.017      URL     [本文引用: 2]

2013年对低温胁迫下烟苗活性氧代谢特性以及活性氧与烟草花芽分化关系进行了研究。结果表明,低温胁迫下,保护酶超氧化物歧化酶(SOD)响应最为迅速,随后过氧化氢酶(CAT)活性逐渐升高,而过氧化物酶(POD)活性响应晚于CAT;CAT在胁迫前中期起保护作用,而POD主要在中后期起保护作用。低温胁迫下,超氧阴离子和过氧化氢都迅速升高,在保护酶系的作用下,活性氧含量呈现动态变化,随着胁迫时间延长,后期超氧阴离子含量升高,过氧化氢含量下降。相关分析表明烟草花芽分化时间与活性氧含量变化有显著相关关系,推测低温胁迫下,烟苗体内活性氧代谢平衡状态的变化可能参与烟草的成花转变,一定程度地促进花芽分化,具体机制有待进一步研究。

段玉琪, 晋艳, 杨宇虹 .

体式显微镜法观察烤烟花芽分化的研究

中国农学通讯, 2011,27(3):143-146.

URL     [本文引用: 2]

The floral bud differentiation processes on apical meristem of flue-cured tobacco induced with low temperature were observed by stereomicroscope. The results showed that this method was simpler and faster than that of scanning electron microscope and paraffin section. It revealed that the stereomicroscope is more superior in showing the spatial characteristics of meristem than that of the scanning electronic microscope method, and the image is more complete. It also validated the uniqueness of the inflorescence initiation of flue-cured tobacco lateral shoot which was observed by scanning electron microscope, and this uniqueness could not be captured or found by paraffin section.

李元元 .

低温诱导烟草早花研究与烟草MADS-box基因的同源克隆

北京:中国农业科学院, 2011.

URL     [本文引用: 1]

烟草是一种重要的叶用经济作物,在烟叶种植过程中易出现早花现象,烟草早花严重影响烟叶产量和质量。低温是诱导烟草早花的重要因素之一,到目前为止,其相关的早花规律研究结果意见不一,其分子机制也一直不清楚。本研究利用低温敏感的烤烟品种NC82,通过不同苗期的低温诱导处理(12℃10 d ),研究该品种在不同苗期对低温的敏感特性;与此同时,采用RT-PCR和RACE等同源克隆技术对NC82中与开花相关的MADS-box家族基因进行同源克隆,并采用半定量RT-PCR和Real-Time PCR技术对克隆得到的基因进行表达分析。该研究具有现实的生产指导意义,也为培育低温不敏感烤烟品种提供很好的功能基因。具体研究结果如下: (1)NC82的低温诱导结果:移栽后第16 d,8叶期低温处理的烟苗首先出现花芽分化迹象,开始由营养生长向生殖生长转变;移栽后第21 d,6叶期和8叶期低温处理的烟苗均出现花芽分化,其对照均在移栽后第26 d左右才出现花芽分化,低温处理分别使其花芽分化提前5 d和8 d;移栽后第31d,6叶期和8叶期低温处理的烟苗提前现蕾,其对照在移栽第36 d左右才现蕾;低温处理和对照烟苗的中心花开放时间相差2~3 d,均在移栽后的第45~49 d; 4叶期低温处理的烟苗则相反,其花芽分化、现蕾和中心花开放分别比对照晚11d、18 d和16 d。所以6叶期和8叶期是NC82低温敏感期,低温能促进早花,但4叶期易受低温伤害,延迟开花;低温处理烟苗的花芽分化到现蕾时间比对照时间长,说明低温延长了花原基的分化过程;而现蕾到开花的天数波动很大,可能受环境影响很大。 (2)烟草MADS-box基因家族中间片段克隆:通过设计简并引物,采用RT-PCR技术从NC82茎尖中成功分离出27条cDNA片段,长度在122~146 bp之间。序列分析表明,其中有16条具有MADS_MEF2类保守结构域,与拟南芥等MADS-box基因家族成员具有很高的同源性,相似性平均能达到76%,并具有15个保守的氨基酸位点,其中7个位点参与蛋白与DNA的相互作用,5个位点具有蛋白二聚化功能。系统发育分析发现这16条保守片段分别和拟南芥9个不同的MADS-box基因家族亚类最近似,其中FLC、STMADS11、TM3和AGL6亚家族具有控制开花时间的功能;AG、AGL2和DEF/GLO亚家族分别属于C/D型、E型和B型花器官同源异型基因;AGL15和AGL12亚家族的基因在茎中也可以表达。 (3)烟草NtFLC的全长克隆及表达分析:利用与拟南芥AtFLC相似性最高的中间片段LyE05设计引物进行3’RACE和5’全长克隆,选取5’完整的片段A5(805 bp)和B1(433 bp),3’RACE的845 bp全长,以及中间保守序列LyE05 (146 bp)进行烟草FLC类基因的全长拼接,得到1174 bp的烟草FLC类全长cDNA。应用NCBI的ORF finder软件,发现其可读框架(ORF)位于172~786位核酸之间,长度615 bp,编码204个氨基酸,该蛋白属于MADS-box基因家族,并含有保守的MADS-box和K-box结构域;系统发育树分析发现其与拟南芥AtFLC具有同源性;半定量RT-PCR的表达分析发现NtFLC主要在茎和茎尖中表达,在花和果实中表达量很少;Real-Time PCR进行准确定量分析表明NtFLC的表达量在低温处理后的茎尖中是降低的。所以推测NtFLC可能与NC82低温敏感,易早花特性相关,在烟草中具有与拟南芥AtFLC相近的功能。

Diego S F, Mayara R B, Daniel G C , et al.

Usual normalization strategies for gene expression studies impair the detection and analysis of circadian patterns

Chronobiology International, 2018,35(3):378-391.

DOI:10.1080/07420528.2017.1410168      URL     PMID:29219623      [本文引用: 1]

Circadian rhythms are controlled by a molecular mechanism that is organized in transcriptional and translational feedback loops of gene expression. Recent studies have been demonstrating the involvement of microRNAs (miRs) in post-transcriptional/translational control of circadian rhythms. In the present study we aimed to analyze the daily variations of miR-16 and miR-181a expression in human... [Show full abstract]

刘秀丽, 招启柏, 袁莉民 , .

烟草花芽分化的形态建成观察

中国烟草科学, 2003(1):9-11.

[本文引用: 1]

杨盛, 郝国伟, 白牡丹 , .

‘玉露香梨’僵芽发生与活性氧代谢的关系

农学学报, 2016,6(2):76-81.

[本文引用: 1]

卢金 .

外源H2O2处理对小麦种子萌发及幼苗生理生化特性的影响

兰州:西北师范大学, 2012.

URL     [本文引用: 1]

活性氧(Reactiveoxygenspecies,ROS)是植物体自身代谢产生的一类物质,主要包括过氧化氢(Hydrogenperoxide,H2O2)、超氧阴离子(O2ˉ)、羟自由基(·OH)、单线态氧(1O2)等。逆境胁迫会导致ROS的的积累,从而氧化损伤细胞组分(破坏蛋白质、膜脂、DNA等)及影响正常代谢。植物体可利用超氧化物歧化酶(Superoxidedismutase,SOD)、过氧化氢酶(Catalase,CAT)、过氧化物酶(Peroxidase,POD)、抗坏血酸过氧化物酶(Ascorbateperoxidase,APX)、谷胱甘肽还原酶(Glutathionereductase,GR)等抗氧化酶来清除多余ROS,使活性氧的产生和清除处于动态平衡,防御ROS积累引起的膜脂过...

李琳, 薛林贵, 张红光 , .

外源过氧化氢对UV-B胁迫下蓝藻生理的影响

生态科学, 2014(4):691-697.

[本文引用: 1]

Singh R, Parihar P .

Reactive oxygen species (ROS):Beneficial companions of plants' developmental processes

Frontiers in Plant Science, 2016,7:1299.

DOI:10.3389/fpls.2016.01299      URL     PMID:5037240      [本文引用: 1]

Reactive oxygen species (ROS) are generated inevitably in the redox reactions of plants, including respiration and photosynthesis. In earlier studies, ROS were considered as toxic by-products of aerobic pathways of the metabolism. But in recent years, concept about ROS has changed because they also participate in developmental processes of plants by acting as signaling molecules. In plants, ROS regulate many developmental processes such as cell proliferation and differentiation, programmed cell death, seed germination, gravitropism, root hair growth and pollen tube development, senescence, etc. Despite much progress, a comprehensive update of advances in the understanding of the mechanisms evoked by ROS that mediate in cell proliferation and development are fragmentry and the matter of ROS perception and the signaling cascade remains open. Therefore, keeping in view the above facts, an attempt has been made in this article to summarize the recent findings regarding updates made in the regulatory action of ROS at various plant developmental stages, which are still not well-known.

Cembrowska L D, Koprowski M, Kepczynski J .

Germination induction of dormant Avena fatua caryopses by KAR1 and GA3 involving the control of reactive oxygen species (H2O2 and O2 -) and enzymatic antioxidants (superoxide dismutase and catalase) both in the embryo and the aleurone layers

Journal of Plant Physiology, 2015,176:169-179.

DOI:10.1016/j.jplph.2014.11.010      URL     PMID:25618514      [本文引用: 1]

Avena fatua L. caryopses did not germinate at 20°C in darkness because they were dormant. However, they were able to germinate in the presence of karrikinolide (KAR1), a key bioactive compound present in smoke, and also in the presence of gibberellin A3 (GA3), a commonly known stimulator of seed germination. The aim of this study was to collect information on a possible relationship between the above regulators and abscisic acid (ABA), reactive oxygen species (ROS) and ROS scavenging antioxidants in the regulation of dormant caryopses germination. KAR1 and GA3 caused complete germination of dormant A. fatua caryopses. Hydrogen peroxide (H2O2), compounds generating the superoxide (O261), i.e. menadione (MN), methylviologen (MV) and an inhibitor of catalase activity, aminotriazole (AT), induced germination of dormant caryopses. KAR1, GA3, H2O2 and AT decreased ABA content in embryos. Furthermore, KAR1, GA3, H2O2, MN, MV and AT increased α-amylase activity in caryopses. The effect of KAR1 and GA3 on ROS (H2O2, O261) and activities of the superoxide dismutase (SOD) and catalase (CAT) were determined in caryopses, embryos and aleurone layers. SOD was represented by four isoforms and catalase by one. In situ localization of ROS showed that the effect of KAR1 and GA3 was associated with the localization of hydrogen peroxide mainly on the coleorhiza. However, the superoxide was mainly localized on the surface of the scutellum. Superoxide was also detected in the protruding radicle. Germination induction of dormant caryopses by KAR1 and GA3 was related to an increasing content of H2O2, O261and activities of SOD and CAT in embryos, thus ROS homeostasis was probably required for the germination of dormant caryopses. The above regulators increased the content of ROS in aleurone layers and decreased the activities of SOD and CAT, probably leading to the programmed cell death. The presented data provide new insights into the germination induction of A. fatua dormant caryopses by KAR1 and also by GA3. In A. fatua, KAR1 or GA3 is included in the induction germination of dormant caryopses through regulation level of ABA in embryos and ROS-antioxidant status both in embryos and aleurone layers.

赵士诚, 孙静文, 马有志 , .

镉对玉米幼苗活性氧代谢、超氧化物歧化酶和过氧化氢酶活性及其基因表达的影响

中国农业科学, 2008,41(10):3025-3032.

Magsci     [本文引用: 1]

<FONT face=Verdana>【目的】研究镉胁迫对玉米(Zea Mays)幼苗活性氧代谢,超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的活性及其基因表达的影响。【方法】用营养液培养的方法研究了不同镉浓度(0﹑5﹑20和100μmol<I>&#</I>8226;L-1)和处理时间(12﹑24﹑48﹑96和168 h)下玉米幼苗内活性氧代谢、SOD和CAT活性及其基因表达的变化。【结果】镉处理后植株内超氧自由基( )产生速率迅速升高,24 h(叶)或48 h(根)后又逐步下降;H2O2随镉浓度和处理时间的增加而大量积累。镉处理的植物SOD活性开始随镉浓度升高,48 h后被消耗和抑制逐步下降,但后期100μmol<I>&#</I>8226;L-1镉处理的活性仍显著高于其它处理;CAT活性除叶中100μmol<I>&#</I>8226;L-1镉处理被抑制降低外均被诱导,开始随镉浓度升高,随后随镉浓度和胁迫时间逐步下降。镉诱导的SOD基因表达与其活性变化相似,而CAT基因表达随镉浓度和处理时间逐步增强,说明在玉米幼苗内镉通过抑制SOD的基因转录抑制其活性,而对CAT,镉胁迫导致其产生了翻译后蛋白修饰。【结论】镉处理诱导了玉米幼苗内活性氧产生、SOD和CAT的活性及基因表达增加,随胁迫的加剧,SOD和CAT的活性和SOD表达被抑制,CAT则产生转录后或翻译后修饰。</FONT>

王鹏程 .

拟南芥活性氧应答基因的转录调控分子机制研究

开封:河南大学, 2010.

DOI:10.7666/d.y1694071      URL     [本文引用: 1]

在高等植物中,活性氧作为重要的信号分子介导了防御反应、程序性死亡、气孔关闭、细胞生长和植物发育调控等许多重要的生理过程。活性氧能够广泛地调控许多基因的表达,暗示了在植物细胞中存在复杂的活性氧应答的转录调控网络,但目前对于这一复杂调控网络中的许多细节仍不清楚。 本研究通过基因芯片技术在基因组水平上系统研究检测了ABA以及H202对拟南芥幼苗基因表达的调控。发现外源H202处理使459个基因的转录水平明显增加,同时使221个基因的表达显著减少,显示H202也是一种重要的转录调节物质,广泛地调控了许多基因的表达。而且ABA和H202对基因表达的调控存在相当大的重叠,它们可能通过一种协同模式调控下游基因的表达。通过生物信息学检索,本研究从活性氧特异应答基因的启动子序列中发现8个潜在的氧化胁迫应答的顺式作用元件。通过基因枪介导的瞬时表达系统检测发现4个保守序列元件在H202、MV等不同种类的活性氧处理下能够诱导下游萤光素酶基因的表达,它们可能作为氧化胁迫应答的元件(ROS responsive element)参与活性氧应答的转录调控。 在上述研究的基础上,发现AP2/EREBP家族转录因子ERF6能够特异地与ROE8结合,共转化ERF6能够显著增强ROE8的转录活性,而且H202处理能够促进ERF6对ROE8的激活效应。进一步的研究发现ERF6能够与MPK3/6发生直接的相互作用,MPK6能够磷酸化ERF6蛋白的Ser266和Ser269残基。ERF6磷酸化位点的突变会引起ERF6亚细胞定位的改变,ERF6DD定位于细胞核中,而非活化状态的ERF6AA主要存在于细胞质中。35S-ERF6WT、35S-ERF6DD超表达转基因植物在萌发和生长早期对H202更加敏感,一些活性氧应答基因的表达也明显增加。免疫共沉淀结果还显示H202和磷酸化能增加ERF6和MPK6复合体的积累。上述结果显示ERF6作为活性氧应答的转录因子,接受活性氧激活的MPK6的调控,介导了活性氧对于基因表达和幼苗生长发育的调控过程。 本研究工作系统比较分析了H202对基因表达的调控作用,通过生物信息学和遗传学分析检测了在活性氧应答基因启动子中存在的氧化胁迫应答元件,并鉴定了其中一个顺式作用元件ROE8的结合蛋白ERF6的功能,发现ROS-MPK6-ERF6途径部分地介导了活性氧对于基因表达的调控过程。这一研究为阐明活性氧应答的转录调控机制提供了重要的基因组学和遗传学线索。

Lu X, Li J, Chen H , et al.

RNA-seq analysis of apical meristem reveals integrative regulatory network of ROS and chilling potentially related to flowering in Litchi chinensis

Scientific Reports, 2017,7(1):10183.

DOI:10.1038/s41598-017-10742-y      URL     PMID:28860553      [本文引用: 1]

Abstract Litchi is an important woody fruit tree. Floral initiation in litchi is triggered by low temperatures. However, defective flowering is a major challenge for litchi production in times of climate change and global warming. Previous studies have shown that the reactive oxygen species (ROS) generated by methyl viologen dichloride hydrate (MV) promotes flowering. In this study, potted trees were transferred to growth chambers for low-temperature (LT), medium-temperature (MT), and high-temperature (HT) treatments. Trees at MT were subjected to ROS treatment to promote flowering, and those at LT were induced to flower. RNA-sequencing was applied to obtain a global transcriptome of the apical meristem and reveal potential gene networks controlling the transformation from vegetative meristems (VM) into inflorescence meristems (IM). We assembled 73,117 unigenes with a mean size of 790 bp and 11741 unigenes were identified as both chilling and ROS responsive genes (CRRGs), of which 48 were identified as flowering-related CRRGs, 59 were plant hormone signal transduction CRRGs, and 146 were plant hormone biosynthesis-related CRRGs. Genes co-expression network analysis indicated inner relationships, suggesting that ROS and chilling promotes the VM to IM transition through a regulatory gene network of transcription factors, hormones, and flowering regulators.

李念祖 .

芥菜开花整合子FLC与SVP相互作用的研究

重庆:西南大学, 2013.

URL     [本文引用: 1]

开花是有性生殖植物从营养生长向生殖生长的关键转折。延缓开花可在生产上避免“未熟抽薹”;提早开花可以加快品种选育进程;调节双亲花期有利于F1制种。芥菜开花时间主要受光照(例如日照长度、日照强度、光周期)、温度等外在环境以及植物生长状况、发育阶段已及遗传因素等内在条件影响。与拟南芥相似,主要有四条途径调节芥菜开花:春化途径、自主途径、光周期途径和赤霉素途径。尽管这四条途径分别有不同的基因网络调控,但最终都汇集到相同的开花途径整合子。目前,拟南芥中以分离出大量与开花时间相关的基因,如FLOWERING LOCUS T(FT)、SUPPRESSOR OF CO OVEREXPRESSION1(SOC1)、FLOWERING LOCUS C (FLC)和SHORT VEGETATIVE PHASE (SVP)等,其中FT与SOC1促进开花,FLC与SVP抑制开花。由于芥菜是重要的十字花科蔬菜作物,属于种子春化类型,在我国南方广泛种植。因此,对芥菜开花时间调控的研究,在生产和实践中具有非常重要的意义。 本实验以芥菜为研究材料,利用已报道的开花基因的保守区域设计简并引物,克隆了两个延迟开花的基因FLC与SVP,并构建了FLC及其截短体、SVP及其截短体的酵母双杂交重组表达载体,筛选与鉴定FLC与SVP相互作用及其蛋白互作的结构域。 1、FLC与SVP的克隆与分析 根据已发布的拟南芥和芸薹属作物中FLC与SVP基因的保守区域设计简并引物,从芥菜cDNA中扩增出FLC与SVP基因的全长cDNA。FLC基因含有625bp,编码197个氨基酸,属于MIKC型MADS蛋白,进化分析表明与芸薹属中甘蓝FLC(AAQ76273)亲缘关系最近。SVP基因含有757bp,编码241个氨基酸,也属于MIKC型MADS蛋白,与芸薹属中油菜SVP(AAQ55451)亲缘关系最近。 2、全长FLC与SVP相互作用的鉴定 利用同源重组技术,构建了FLC与SVP的酵母表达载体pGADT7FLC\pGADT7SVP\pGBKT7FLC和pGBKT7SVP。利用醋酸锂转化法将重组酵母质粒转化到感受态酵母中,得到酵母转化子Y187(pGADT7FLC)、Y187(pGADT7SVP)、 Y2HGold (pGBKT7FLC)和Y2HGold (pGBKT7SVP),经过不同缺陷型培养基鉴定,发现无自激活和毒性现象。融合的二倍体酵母Y187(pGADT7FLC)×Y2HGold (pGBKT7SVP)和Y187(pGADT7SVP)×Y2HGold (pGBKT7FLC)均能在选择型培养基SD/-Leu/-Trp/AbA (DDO/A)、SD/-Ade/-His/-Leu/-Trp (QDO)、 SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA (QDO/X/A)上正常生长。结果表明,克隆到的芥菜FLC与SVP蛋白能够相互接合,为深入研究FLC-SVP作用位点建立一个技术平台。 .3、FLC-SVP相互作用区域的筛选与鉴定 从全长的FLC和SVP亚克隆了5个FLC截短体(FLC1~5)和5个SVP截短体(FLC1~5)。FLC1~5和SVP1~5编码蛋白的结构域均分别为MI、MIK、K、IKC和KC,并构建酵母重组表达质粒pGADT7SVP1~5和pGBKT7FLC1~5,并转化对应的酵母菌。二倍体酵母Y187(pGADT7SVP2-5)×Y2HGold (pGBKT7FLC)和Y187(pGADT7SVP)×Y2HGold (pGBKT7FLC2-5),在QDO/X/A培养基上长出蓝色菌落,同时激活了酵母的报告基因AUR1-C, HIS3、ADE2、MEL1。由此表明,FLC与SVP2-5能够相互作用,同时SVP与FLC2~5也能互作,从而暗示SVP与FLC的K域是介导FLC-SVP二聚化的核心部位。进一步研究发现,Y187(pGADT7FLC3)×Y2HGold (pGBKT7SVP3)和Y187(pGADT7SVP3) xY2HGold (pGBKT7FLC3)二倍体酵母,在QDO/X/A培养基上长出蓝色菌落,表明FLC3与SVP3能互作,进一步证实了K域是FLC-SVP相互作用的核心区域。

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