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矮牵牛花瓣衰老和逆境胁迫响应相关NAC基因的鉴定与分析

杨应杰 张付昆 穆静怡 付鲁峰 陈倬 李华 关夏玉 吕培涛

杨应杰,张付昆,穆静怡,等. 矮牵牛花瓣衰老和逆境胁迫响应相关NAC基因的鉴定与分析 [J]. 福建农业学报,2024,39(6):700−710 doi: 10.19303/j.issn.1008-0384.2024.06.009
引用本文: 杨应杰,张付昆,穆静怡,等. 矮牵牛花瓣衰老和逆境胁迫响应相关NAC基因的鉴定与分析 [J]. 福建农业学报,2024,39(6):700−710 doi: 10.19303/j.issn.1008-0384.2024.06.009
YANG Y J, ZHANG F K, MU J Y, et al. Identification and Analysis of NAC Related to Petal Senescence and Stress Responses of Petunia [J]. Fujian Journal of Agricultural Sciences,2024,39(6):700−710 doi: 10.19303/j.issn.1008-0384.2024.06.009
Citation: YANG Y J, ZHANG F K, MU J Y, et al. Identification and Analysis of NAC Related to Petal Senescence and Stress Responses of Petunia [J]. Fujian Journal of Agricultural Sciences,2024,39(6):700−710 doi: 10.19303/j.issn.1008-0384.2024.06.009

矮牵牛花瓣衰老和逆境胁迫响应相关NAC基因的鉴定与分析

doi: 10.19303/j.issn.1008-0384.2024.06.009
基金项目: 福建农林大学园艺学院青年学术骨干培养基金项目(722022011);福建省高原学科建设项目(102/71201801101)
详细信息
    作者简介:

    杨应杰(1996 —),男,硕士研究生,主要从事花卉与景观园艺研究,E-mail:hnyingjieyang@163.com

    通讯作者:

    关夏玉(1984—),女,博士,高级实验师,主要从事观赏园艺品质生物学研究,E-mail:gxy302@126.com

    吕培涛(1983 —),男,博士,教授,主要从事园艺作物品质生物学研究,E-mail:ptlv@fafu.edu.cn

  • 中图分类号: S681.6

Identification and Analysis of NAC Related to Petal Senescence and Stress Responses of Petunia

  • 摘要:   目的  NAC(NAM, ATAF and CUC)参与植物生长发育和多种逆境胁迫响应过程的调控。本文旨在鉴定和研究对矮牵牛生长发育和逆境胁迫响应的关键NAC成员,为优质抗逆矮牵牛育种提供基因资源。  方法  以腋生矮牵牛(Petunia axillaris)基因组为参考基因组,利用矮牵牛花器官衰老过程、烟草脆裂病毒(Tobacco rattle virus, TRV)侵染、低磷、低温、NaCl、铜离子和干旱胁迫处理后的转录组数据,分析矮牵牛NAC基因(PaNACs)差异表达情况,并对差异表达PaNACs的启动子顺式作用元件及转录因子结合位点进行分析。利用实时荧光定量PCR验证了部分差异表达PaNACs在矮牵牛花衰老过程中的表达情况,并预测了差异表达PaNACs编码蛋白的潜在靶基因。  结果  鉴定的131个PaNAC基因中,59个(45.04%)被鉴定为花器官衰老和逆境胁迫响应过程中的差异表达基因。PaNAC72、PaNAC22、PaNAC29、PaNAC40、PaNAC2、PaNAC90、PaNAC83、PaNAC56、PaNAC36PaNAC35在至少3个生物学过程响应中差异表达显著,其中拟南芥衰老关键基因AtNAP的直系同源基因PaNAC29在花器官衰老过程和低温、低磷、铜离子胁迫逆境处理中显著上调表达;PaNAC72在除受铜离子胁迫外的所有6种处理中表达差异显著;PaNAC22在花器官衰老过程和低温和低磷胁迫中上调表达,在铜离子和干旱逆境下调表达。启动子分析结果显示这10个PaNAC启动子区域存在多种逆境胁迫响应相关元件,且大量响应衰老和逆境胁迫的差异表达基因的启动子区域存在NAC的结合位点。  结论  PaNACs广泛参与矮牵牛生长发育及逆境胁迫响应,其中PaNAC29可能是花衰老关键的正调控因子,PaNAC72广泛响应多种逆境胁迫。
  • 图  1  拟南芥AtNAC和矮牵牛PaNAC系统发育树

    Figure  1.  Phylogenetic tree for Arabidopsis AtNACs and petunia PaNACs

    图  2  矮牵牛花衰老过程中PaNACs表达情况

    A:火山图展示矮牵牛NAC家族成员在花衰老过程中的差异表达基因;B:热图展示矮牵牛花衰老过程中表达显著变化的NAC基因;C:D0和D4时期矮牵牛花表型;D:PaNAC29PaNAC72的RT-qPCR验证结果;***: P< 0.001。

    Figure  2.  Expressions of PaNACs during petunia flower senescence

    A: Volcano diagram showing differentially expressed PaNACs in flower senescence; B: heatmap showing NACs with significant changes in expression during petunia flower senescence; C: petunia flower phenotypes on D0 and D4; D: RT-qPCR validation results of PaNAC29 and PaNAC72, respectively; ***: P<0.001.

    图  3  矮牵牛叶在烟草脆裂病毒(TRV)侵染过程中PaNACs表达情况

    A:矮牵牛NAC家族成员在TRV病毒侵染过程中的差异表达基因火山图;B:矮牵牛在TRV病毒侵染过程中差异表达NAC基因热图;S0、S3、S6分别为病毒侵染后第0、3、6天。

    Figure  3.  Expressions of PaNACs in petunia leaves under TRV infestation

    A: Volcano diagram showing differentially expressed PaNACs in TRV-infested petunia; B: heatmap showing NACs with significant changes in expression during TRV infestation in petunia. S0, S3, and S6: 0, 3, and 6 d after viral inoculation, respectively.

    图  4  多种处理条件下矮牵牛PaNACs表达情况

    A~E:气泡图展示矮牵牛NAC家族成员在低温(A)、低磷(B)、铜离子(C)、NaCl(D)和干旱(E)等非生物胁迫下的表达情况;F:韦恩图展示矮牵牛NAC家族成员在花衰老和非生物胁迫过程中的差异表达基因;G:矮牵牛花衰老和非生物胁迫中参与多个过程的PaNAC

    Figure  4.  PaNACs expressions in petunia under various conditions

    A–E: bubble plots demonstrating expressions of PaNACs under abiotic stresses, such as cold (A), low phosphorus (B), copper ions (C), NaCl (D), and drought (E); F: Wayne diagram showing differentially expressed PaNACs during flower senescence and under abiotic stresses; G: PaNACs involved in multiple processes in petunia flower senescence and abiotic stresses.

    图  5  10个在花衰老和胁迫差异表达的PaNACs的启动子顺式作用元件及转录因子结合位点预测结果

    A:10个在花衰老过程和胁迫处理下差异表达的PaNACs启动子顺式作用元件预测结果;B:10个在花衰老和胁迫差异表达的PaNAC启动子的转录因子结合位点预测。

    Figure  5.  Cis-acting elements and transcription factor binding sites in promoters of 10 PaNACs with differential expressions during flower senescence and under stresses

    A: Cis-acting elements in promoters of 10 PaNACs showing differential expression during flower senescence and in response to stresses; B: transcription factor binding site prediction for promoters of 10 differentially expressed PaNACs during flower senescence and in response to stresses.

    图  6  荧光定量PCR测定PaNAC在花衰老过程中的表达量

    ns表示P> 0.05,*表示P < 0.05 ,**表示P < 0.01 ,***表示P < 0.001。

    Figure  6.  Expressions of PaNACs during flower senescence by qRT-PCR

    ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001.

    图  7  矮牵牛衰老和逆境胁迫中的差异表达基因及PaNAC的潜在靶基因的GO富集

    A:矮牵牛花衰老和逆境胁迫中的差异表达基因数量;B:差异表达基因中PaNAC的潜在靶基因的GO富集。

    Figure  7.  Differentially expressed genes in petunia senescence and under stresses and GO enrichment of potential target genes of PaNAC

    A: number of differentially expressed genes in petunia flower senescence and stresses; B: GO enrichment of differentially expressed genes for potential target genes of PaNAC.

    表  1  矮牵牛花衰老及各种胁迫处理

    Table  1.   Petunia flower senescence and various stress treatments

    胁迫
    Stress
    处理方法
    Treatment
    品种/组织
    Cultivar/tissue
    数据来源
    Data source
    花衰老
    Flower senescence
    采样时间点:第0天(D0),花朵开放但在花药裂开之前;第4天(D4),花冠在尖端边缘显示枯萎迹象。 ‘米切尔二倍体’腋生矮牵牛/花瓣 PRJNA417209[12]
    病毒胁迫
    Virus stress
    用100 mmol·L−1酸盐缓冲液均质化的TRV (PPK20) 感染性汁液侵染,在侵染后第0天(S0)、3天(S3)和6天(S6)取样。 ‘蓝色好时’腋生矮牵牛/叶 PRJNA693880[13]
    冷胁迫
    Cold stress
    分别于4 °C低温处理后1、3、6、12 h收集叶片。 ‘超越’腋生矮牵牛/叶 PRJNA640832[14]
    低磷胁迫
    Low phosphorus
    切除来自节点2(侧芽)和7(顶芽)的腋芽;两种处理:正常磷(250 μmol·L−1)和低磷(5 µmol·L−1)。 ‘V26自交系’腋生矮牵牛/腋芽 PRJNA997338[15]
    NaCl胁迫
    NaCl stress
    正常植株用Hoagland溶液进行培养;NaCl处理组用含150 mmol·L−1 NaCl的改良Hoagland溶液进行处理,在处理后0、6、24 h取叶片。 ‘米切尔二倍体’腋生矮牵牛/叶 PRJNA381775[16]
    铜离子胁迫
    Cu stress
    正常营养液培养28 d后,将植物转移至含有40 μmol·L−1 CuSO4(+Cu)的营养液中直至开花;正常植株始终用正常营养液培养。收集第7阶段(花药裂开)花瓣。 ‘米切尔二倍体’腋生矮牵牛/花瓣 PRJNA774370[17]
    干旱胁迫
    Drought stress
    对照组每天用100 mg·L−1氮灌溉直至试验结束,胁迫组5 d不浇水。并在第5天收集叶。 ‘米切尔二倍体’腋生矮牵牛/叶 PRJNA680631[18]
    下载: 导出CSV

    表  2  实时荧光定量PCR引物信息

    Table  2.   Oligonucleotide primers used for qRT-PCR analysis

    基因名称
    Gene
    上游引物
    Forward primer
    下游引物
    Reverse primer
    退火温度
    Annealing temperature/ ℃
    PaNAC2 CTAATGTCGACCGCTCTGCT CATCGATTGTGGCCTTGGTG 57.34/57.03
    PaNAC22 ATAGCCAACGTGACCGGAAG AGAGTAGTGAGGGTCGGTCC 57.65/58.56
    PaNAC29 TCGGACCTTCCTCCAGGATT TATCGGTGCCTGTAGCCTTC 57/58
    PaNAC35 GGATGACAGAAGCAGCAACG GTTCCCAAGGGTCATAGCGA 56.89/57.23
    PaNAC36 TGGCAACAATTGGCGAGAGA ACCCAATCAGTCTTGGAGCC 57.23/57.56
    PaNAC40 GTCTCCAGTGGGCCTGAATC TCAACCAGCTTGCTGAACCA 58.49/57.19
    PaNAC72 TGTGTCACAGGGTACTCAAGC ACCGAATACCAAACGGGTCA 57/56.4
    Actin7 TGCTGATCGTATGAGCAAGGAA GGTGGAGCAACAACCTTAATCTTC 56/56
    下载: 导出CSV
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  • 收稿日期:  2024-03-25
  • 修回日期:  2024-06-15
  • 网络出版日期:  2024-08-15
  • 刊出日期:  2024-06-28

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