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蒺藜苜蓿生物钟基因MtTOC1a的克隆及功能验证

段婷婷, 杨明康, 黄可, 黄巍

段婷婷, 杨明康, 黄可, 等. 蒺藜苜蓿生物钟基因MtTOC1a的克隆及功能验证[J]. 华南农业大学学报, 2023, 44(5): 803-809. DOI: 10.7671/j.issn.1001-411X.202211001
引用本文: 段婷婷, 杨明康, 黄可, 等. 蒺藜苜蓿生物钟基因MtTOC1a的克隆及功能验证[J]. 华南农业大学学报, 2023, 44(5): 803-809. DOI: 10.7671/j.issn.1001-411X.202211001
DUAN Tingting, YANG Mingkang, HUANG Ke, et al. Cloning and functional verification of circadian clock gene MtTOC1a in Medicago truncatula[J]. Journal of South China Agricultural University, 2023, 44(5): 803-809. DOI: 10.7671/j.issn.1001-411X.202211001
Citation: DUAN Tingting, YANG Mingkang, HUANG Ke, et al. Cloning and functional verification of circadian clock gene MtTOC1a in Medicago truncatula[J]. Journal of South China Agricultural University, 2023, 44(5): 803-809. DOI: 10.7671/j.issn.1001-411X.202211001

蒺藜苜蓿生物钟基因MtTOC1a的克隆及功能验证

基金项目: 广东省自然科学基金(2021A1515012148,2019A1515012009);岭南现代农业科学项目(NZ2021001)
详细信息
    作者简介:

    段婷婷,硕士研究生,主要从事植物生物钟节律调控机制相关研究,E-mail: t_tingduan@163.com

    通讯作者:

    黄 巍,教授,博士,主要从事植物生物钟节律调控机制相关研究,E-mail: weihuang@scau.edu.cn

  • 中图分类号: Q78;S542

Cloning and functional verification of circadian clock gene MtTOC1a in Medicago truncatula

  • 摘要:
    目的 

    分析蒺藜苜蓿Medicago truncatula生物钟基因MtTOC1a的蛋白结构,探究MtTOC1a在生物钟系统中的生物学功能,比较其与拟南芥Arabidopsis thalianaAtTOC1功能相似性和差异性。

    方法 

    通过生物信息学分析,在全基因组范围内鉴定了TOC1在蒺藜苜蓿中的同源基因。构建MtTOC1a基因的表达载体,利用农杆菌介导法引入到拟南芥野生型Col、及相应的功能丧失突变体toc1-2中,进行遗传互补分析。

    结果 

    MtTOC1a和MtTOC1b均具有保守的功能结构域和三维结构。遗传分析表明,在早期光形态建成中,外源转化的MtTOC1a完全恢复了toc1-2的下胚轴伸长表型,但对toc1-2的提前开花表型没有显著影响。在引入CAB::LUC报告基因的株系中,外源转化MtTOC1a在连续光照下使短周期突变体toc1-2的近日节律周期延长,但仍不能完全恢复至野生型水平。

    结论 

    MtTOC1a和拟南芥AtTOC1的功能存在相似性,但在不同的下游调控途径中所扮演的角色存在差异。本研究结果为进一步探索MtTOC1a基因的功能,利用MtTOC1a基因改造苜蓿的重要性状提供了理论依据。

    Abstract:
    Objective 

    The goal of this study is to analyze the protein structure of Medicago truncatula MtTOC1a, explore the biological function of MtTOC1a in the circadian clock system, and compare its similarities and differences in function with its ortholog AtTOC1 in Arabidopsis thaliana.

    Method 

    The orthologous genes of TOC1 in Medicago were identified through bioinformatics analysis, the expression vector of MtTOC1a gene was constructed and introduced into Arabidopsis wild-type Col and the corresponding loss-of-function mutant toc1-2 by Agrobacterium mediated method for genetic complementation analysis. Both MtTOC1a and MtTOC1b have conserved functional domains and protein structures. The genetic analysis indicated that during early photomorphogenesis, exogenously transformed MtTOC1a fully restored the hypocotyl elongation phenotype of toc1-2, but had no significant effect on the premature flowering phenotype of toc1-2. In the CAB::LUC reporter lines, MtTOC1a lengthened the period of the short period mutant toc1-2 under continuous light conditions, yet the mutant could not fully recover to the wild-type level.

    Conclusion 

    MtTOC1a and AtTOC1 have similar functions, but their roles in drownstream pathways are still different. The results provide a theoretical basis for further exploring the function of MtTOC1a gene and using MtTOC1a gene to modify the important traits in Medicago.

  • 普通大蓟马Megalurothrips usitatus又名豆大蓟马、豆花蓟马,隶属于缨翅目蓟马科大蓟马属,主要分布于澳大利亚、马来西亚、斯里兰卡、菲律宾、斐济、印度、日本等[1-3],在我国海南、台湾、广东、广西、湖北、贵州、陕西等地也均有发生为害[4-5]。据报道,该虫有28种寄主,其中16种为豆科植物,目前它已成为危害华南地区豆科作物的主要害虫[6-9],田间调查和室内试验均表明豇豆为其嗜好寄主[10-11]。普通大蓟马主要以锉吸式口器取食豇豆幼嫩组织的汁液,可造成叶片皱缩、生长点萎缩、豆荚痂疤等,严重影响豇豆品质[12-13]。此外,该虫体积小、发生量大、隐秘性强,大部分时间都躲在花中取食,从豇豆苗期至采收期均可为害[14-15],以上特点均增加了农户的防治难度。当其为害严重时,农户只能增加施药频率和施药量,这也导致该虫对多种常用化学农药产生了严重的抗药性[16-17]

    目前关于普通大蓟马的研究主要集中在生物学特性[18]及综合防治技术[19-20]等层面,随着抗药性的不断发展与研究的不断深入,从分子层面解析普通大蓟马的抗药性机制和寄主选择机制等以寻求新型绿色防控方法势在必行,室内种群的大规模饲养是展开这些研究的基础。化蛹基质作为影响昆虫种群规模的关键因子,韩云等[21]曾指出普通大蓟马在含水量(w)为15%的砂壤土中羽化率显著高于砂土、壤土和黏土,但不适用于室内大规模饲养,因为实际应用中,存在土壤类型无法明确区分、配制砂壤土会增加人工饲养的工作量等问题。土壤以外的其他基质对普通大蓟马化蛹的适合度鲜见研究报道。

    本研究以普通大蓟马为试验对象,室内观测其在沙子、蛭石和厨房用纸3种基质及无基质条件下的羽化规律,分析该虫对不同化蛹基质的适合度,以期为普通大蓟马的室内大规模饲养提供基础资料,为该虫的综合治理提供理论依据。

    普通大蓟马于2017年采自广东省广州市增城区朱村豇豆田,采回后在RXZ-500C型智能人工气候箱(宁波江南仪器厂)内用豇豆豆荚饲养,饲养条件为温度(26±6) ℃,光照周期12 h光∶12 h暗,相对湿度(70±5)%。室内饲养多代后,选取发育一致的老熟2龄若虫(以体色变为橙红色为标准)进行室内试验。

    供试基质包括沙子、蛭石、锯末和厨房用纸,并以无基质作为空白对照。试验前将沙子、蛭石和锯末置于DHG-9140型电热恒温鼓风干燥箱(上海精宏实验设备有限公司)中105 ℃恒温烘烤6 h备用。

    首先称取过筛烘干后的沙子50 g 3组,分别加入2.5、3.5和4.5 mL蒸馏水,充分混匀,配制成含水量(w)分别为5%、7%和9%的沙子化蛹基质;称取过筛烘干后的蛭石10 g 3组,分别加入10.0、12.5和15.0 mL蒸馏水,充分混匀,配制成含水量(w)分别为20%、25%和30%的蛭石化蛹基质;称取过筛烘干后的蛭石10 g 3组,分别加入12.5、15.0和17.5 mL蒸馏水,充分混匀,配制成含水量(w)分别为25%、30%和35%的锯末化蛹基质。将以上基质分别转移至350 mL玻璃组培瓶内,基质深度均为5 cm,将厨房用纸对折成合适大小后平铺在组培瓶底部作为基质。在所有基质上放置纱网,再加入1根新鲜的豇豆豆荚(长度约4~5 cm),分别接入50头普通大蓟马老熟2龄若虫,用250目纱布封口后置于人工气候箱中饲养,每日观察并记录成虫羽化数量。每个处理设6次重复。设置不加入任何化蛹基质的空白对照。

    含水量的测定方法按以下公式[22]进行:

    含水量=实际含水质量/烘干后基质质量×100%。

    运用SPSS 24.0软件进行试验数据处理分析,不同基质及含水量对普通大蓟马羽化率、蛹历期和性比(雄性∶雌性)的影响采用单因素方差分析,并运用Duncan’s法检验差异显著性。

    普通大蓟马在不同基质中的羽化率、蛹历期和性比具有显著差异(图1)。由图1A可知,普通大蓟马在厨房用纸中的羽化率显著高于其他基质,为54.33%,其次为含水量5%(w)的沙子,羽化率为44.67%;锯末最不适宜于普通大蓟马羽化,在含水量(w)为25%、30%、35%的锯末中普通大蓟马的羽化率分别为10.33%、5.33%、16.67%,显著低于空白对照与其他基质。

    图  1  不同基质对普通大蓟马羽化率、发育历期和性比(雄性∶雌性)的影响
    1~3分别为含水量(w)为5%、7%和1%的沙子,4~6分别为含水量(w)为20%、25%和30%的蛭石,7~9分别为含水量(w)为25%、30%和35%锯末,10:厨房用纸,11:无基质;各图中的不同小写字母表示差异显著(P<0.05,Duncan’s法)
    Figure  1.  Effects of different substrates on eclosion rate, pupa developmental period and male-female ratio of Megalurothrips usitatus
    1: Sand with 5% moisture, 2: Sand with 7% moisture, 3: Sand with 10% moisture, 4: Vermiculite with 20% moisture, 5: Vermiculite with 25% moisture, 6: Vermiculite with 30% moisture, 7: Sawdust with 25% moisture, 8: Sawdust with 30% moisture, 9: Sawdust with 35% moisture, 10: Kitchen paper, 11: No substrate; Different lowercase leters in the same figure indicated significant difference among different substrate (P<0.05, Duncan’s method)

    图1B可知,普通大蓟马在含水量5%(w)的沙子中蛹的发育历期最短,为5.29 d,其次为含水量7%(w)的沙子,为6.01 d,在其他基质中的蛹期则无显著差异,在6.14~7.16 d。

    图1C可知,普通大蓟马在含水量30%(w)的蛭石中性比最高,为0.60,含水量10%(w)的沙子和30%(w)的蛭石性比相对较低,分别为0.12和0.06,在其他基质中性比无显著差异。

    表1数据可知,沙子含水量(w)为5%时普通大蓟马羽化最早,始于第2天;其次为蛭石,羽化始于第4天,其他条件下羽化均始于第3天;以锯末为基质时羽化最晚,始于第5天。沙子含水量(w)为5%和厨房用纸条件下,羽化高峰出现在第5天,羽化率分别为21%和22.67%;次高峰在第6天,羽化率分别为14.33%和21%。沙子含水量(w)为9%、锯末以及空白对照下羽化高峰出现在第7天,其他条件下羽化高峰均出现在第6天。不同基质类型及含水量条件下,普通大蓟马的羽化均结束于第8天或第9天,与不同基质培养条件下普通大蓟马蛹期之间的差异相对应。

    表  1  不同基质对普通大蓟马逐日羽化率的影响1)
    Table  1.  Effects of differents substrates on daily eclosion rate of Megalurothrips usitatus %
    t/d 沙子含水量(w) Water content in sand 蛭石含水量(w) Water content in vermiculite
    5% 7% 9% 20% 25% 30%
    1 0 0 0 0 0 0
    2 1.67±0.42c 0 0 0 0 0
    3 1.00±1.68c 0 0 0 0 0
    4 1.33±0.67c 5.33±0.33c 0.33±0.33b 0 0 0
    5 21.00±3.82a 5.33±2.17b 2.67±1.91b 3.00±2.30bc 10.33±3.48ab 0.33±0.33b
    6 14.33±4.66b 17.33±1.76a 2.67±1.91b 11.67±2.09a 14.67±3.33a 7.67±2.22a
    7 2.33±0.80c 5.00±0.85b 8.67±1.84a 6.33±2.28b 7.67±1.74bc 6.67±1.52a
    8 0.67±0.42c 0.67±0.67c 0.67±0.42b 4.00±1.35bc 4.00±1.37cd 1.67±0.94b
    9 0 0 0.33±0.33b 0.67±0.42b 0 0.67±0.42b
    10 0 0 0 0 0 0
    下载: 导出CSV 
    | 显示表格

    化蛹基质的类型对普通大蓟马化蛹具有一定影响,本研究发现锯末和蛭石不适宜于普通大蓟马化蛹,锯末和蛭石不同含水量条件下大蓟马的羽化率都显著低于空白对照。有研究指出土壤中砂土含量低于30%时,蓟马若虫不能化蛹[23],蓟马在砂壤土中的羽化率也显著高于砂土、黏土、壤土等单一土壤[21]

    化蛹基质的含水量对普通大蓟马化蛹具有显著影响,本研究发现当沙子含水量(w)为5%时,羽化率仅次于厨房用纸,高达44.67%,与孟国玲等[23]关于豆带蓟马Taenithripsglycines在含水量(w)为5.7%时羽化率最高(43.63%)的报道相对一致。韩云等[21]研究发现普通大蓟马在含水量(w)为15%的砂壤土中羽化率最高,为52.08%,而土壤含水量(w)5%时羽化率仅为6.67%。这与本研究结果不符,究其原因可能是不同类型的基质吸水力与保水力不同,导致在相同的绝对含水量下湿度有差异。此外,有研究曾指出高含水量不利于蓟马化蛹[24],这与本研究结果相一致,沙子含水量(w)5%时的羽化率显著高于含水量(w)7%和10%。

    在本研究中,成虫性比普遍低于1∶1,含水量(w)30%的蛭石羽化性比最高,为0.6,含水量(w)30%锯末最低,为0.06,其他处理的性比无显著差异,为0.12~0.48。张念台[8]和谭柯[24]在田间调查的结果也显示其成虫性比低于1∶1,后代总是偏于雌性,谭柯[24]则表示后代偏雌性可能是蓟马暴发的原因之一。这与本研究结果相一致,后代偏于雌性。

    本研究发现普通大蓟马在厨房用纸中的羽化率最高,蛹发育历期与其他基质相比无明显差异,且以厨房用纸为化蛹基质时,可以清楚地观察到普通大蓟马蛹期的形态特征变化,可以随时根据试验需求收集不同时期的若虫或成虫。虽然沙子含水量(w)5%时蛹发育历期最短且羽化率也较高,但蓟马一旦入土化蛹便无法继续观察形态或收集虫体。因此,本试验条件下,厨房用纸是最适合室内普通大蓟马大量饲养的化蛹基质。

  • 图  1   蒺藜苜蓿和拟南芥PRR家族基因的系统发育树分析和保守结构域预测

    Figure  1.   Phylogenetic tree analysis and domain prediction of PRR family genes in Arabidopsis thaliana and Medicago truncatula

    图  2   AlphaFold项目所预测的MtTOC1a、MtTOC1b和AtTOC1的蛋白三维结构

    PR结构域与置信得分(pLDDT)≥90的深蓝色区域重合,呈多股α螺旋和β折叠构成的桶状结构;CCT结构域与90> pLDDT≥50的浅蓝色/黄色区域重合,由两条α螺旋构成类似剪刀状的结构

    Figure  2.   3D protein structures of MtTOC1a, MtTOC1b and AtTOC1 predicted by the AlphaFold project

    The PR domain overlaps with the dark blue region with a confidence score of (pLDDT) ≥90, showing a barrel-like structure composed of multiple strands of α helices and β folds; The CCT domain overlaps with the light blue/yellow region with 90> pLDDT≥50, forming a scissor-like structure with two α helices

    图  3   MtTOC1a表达载体的构建

    A:MtTOC1a互补表达载体的T-DNA区域示意图;B:苜蓿MtTOC1a基因的克隆;C:MtTOC1a载体的菌落PCR结果,“*”表示阳性菌落

    Figure  3.   Construction of MtTOC1a expression vector

    A: Schematic diagram of the T-DNA region of MtTOC1a expression vector; B: Cloning of MtTOC1a gene in Medicago; C: Colony PCR results of MtTOC1a expression vector, “*” indicates positive colony

    图  4   MtTOC1a相关转基因植株表型

    A:苗龄7 d的下胚轴长度表型;B:在短日照条件下的开花时间表型

    Figure  4.   Phenotypes of transgenic plants with MtTOC1a

    A: The hypocotyl length phenotypes of 7-day-old seedlings; B:The flowering time phenotypes under short-day conditions

    图  5   MtTOC1a相关转基因植株的表型量化分析

    A:苗龄7 d的下胚轴长度,n≥30;B:在短日照条件下植株抽薹时莲座叶的数量, n≥15;柱子上方的不同小写字母表示差异显著(P<0.05,LSD法)

    Figure  5.   Quantification analysis of the phenotypes of transgenic plants with MtTOC1a

    A: The hypocotyl lengths of 7-day-old seedlings, n≥30; B: The number of rosette leaves during bolting of plants under short-day conditions, n≥15; Different lowercase letters on bars indicate significant differences (P<0.05, LSD test)

    图  6   MtTOC1a相关转基因植株的近日节律周期分析

    A:持续光照条件下的MtTOC1a相关转基因拟南芥植株生物发光节律,n≥16,图中所有植株均带有CAB::LUC荧光素酶报告基因,括号内表示相应植株的近日节律周期,浅灰色表示主观黑夜; B: A图中植株的近日节律周期和相对振幅误差的量化,相对振幅误差数值越小表示植株的节律性越强

    Figure  6.   Circadian rhythm analysis of transgenic plants with MtTOC1a

    A: The bioluminescence rhythm of transgenic Arabidopsis plants with MtTOC1a under continuous light condition, n≥16, all plants in the figure carried a CAB:: LUC reporter gene, the daily rhythm cycle of corresponding plants was indicated in parentheses, light gray represents subjective night; B: Quantification of the circadian rhythm period and relative amplitude error of plants in A, and the smaller the relative amplitude error value, the stronger the rhythmicity of the plant

    表  1   克隆载体的构建引物序列

    Table  1   The primers used for cloning vector construction

    基因
    Gene
    引物名称
    Primer name
    引物序列 (5′→3′)
    Primer sequence
    MtTOC1a MtTOC1a-F CTGATCATGGAGAGTGAAGGGTTTGATTTG
    MtTOC1a-R TTGCTCACCATAGCATCCCTCGGAGAGTAATCTC
    AtTOC1 AtTOC1pro-F CTCGGTACCCGGGGATCCGAGATCGCTCGGCTCAACAA
    AtTOC1pro-R TTCACTCTCCATGATCAGATTAACAACTAAACCCACACA
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出版历程
  • 收稿日期:  2022-10-31
  • 网络出版日期:  2023-11-12
  • 发布日期:  2023-08-27
  • 刊出日期:  2023-09-09

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