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首页 > 过刊浏览>2019年第20卷第4期 >1041-1051. DOI:10.13430/j.cnki.jpgr.20181205001
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13个苹果NAC转录因子基因的克隆与表达分析
作者:
作者单位:

1.山东省果树研究所，山东泰安 271000;2.中国农业科学院果树研究所，辽宁兴城 125100;3.山东农业大学园艺科学与工程学院，山东泰安 271018

基金项目:

国家自然科学基金（31501742）；山东省农业良种工程（2016LZGC034）；山东省农科院农业科技创新工程（CXGC2016A03）


Cloning, Sequence and Expression Analysis of Thirteen NAC Transcription Factors (MdNACs) in Apple
Author:
Affiliation:

1.Shandong Institute of Pomology, Shandong, Taian 271000;2.Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Liaoning, Xingcheng 125100;3.College of Horticulture Science and Engineering, Shandong Agricultural University, Shandong, Taian 271018

Fund Project:

National Natural Science Foundation of China（31501742）；Shandong Agricultural Good Cultivar Project（2016LZGC034）；Agricultural Science and Technology Innovation Project of SAAS（CXGC2016A03）

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    摘要:

    利用同源比对和 RT-PCR 技术，从‘紫弘富士’苹果（Malus domestica cv. Zihong Fuji）中克隆了 13 个 NAC 转录因 子基因：MdNAC40-45，MdNAC47-53（GenBank 登录号为 MG099877-MG099882，MG099884-MG099890）。序列分析表明， 其开放阅读框（open reading frame，ORF）分别为 1 470、1 065、912、885、948、1 041、1 182、639、705、1 830、1 980、 747 和1 137 bp。进化分析表明，MdNAC41属于AtNAC3组，MdNAC40、MdNAC42-43 和MdNAC45-53 属于NAM组，MdNAC44 属于 VND 组。亚细胞定位预测结果显示，MdNAC40-45、MdNAC47-50 和 MdNAC52-53 可能定位于细胞核中；MdNAC51 可能定位在细胞质或细胞核中。Array 结果显示，13 个 MdNAC 基因在 16 个被检测组织中均有不同程度的相对表达水平。 RNA-seq 结果显示，MdNAC43、MdNAC45、MdNAC47、MdNAC49 和 MdNAC51 的表达明显受斑点落叶病菌（Alternaria alternata apple pathotype, AAAP）侵染的诱导。实时荧光定量 PCR 分析表明，在 150 mmol/L NaCl 处理下，‘嘎啦’组培苗中 MdNAC45、 MdNAC47 和 MdNAC52 的转录水平受到诱导，而 MdNAC40、MdNAC48 和 MdNAC51 的转录水平下调；在 300 mmol/L 甘露醇 处理下，MdNAC50 转录水平受到诱导，MdNAC41 转录水平下调。这表明 MdNAC 基因多呈组成型表达，并受斑点落叶病菌 （AAAP）、NaCl 和甘露醇诱导或下调，可能参与调控苹果生长发育和逆境胁迫等过程。

    Abstract:

    NAC transcription factors are one of plant-specific transcription factors that participate in the regulation of plant growth and development as well as the responses to environmental stress. In relative to the in-depth study of NAC transcription factors in model plants Arabidopsis and rice, there are few related studies on NAC transcription factors in apple. In this study, to explore the role of NAC transcription factors in growth and development, as well as biotic and abiotic stress response of apple (Malus×domestica Borkh.), full-length coding sequences of 13 MdNAC genes were isolated by search for sequence homolog and RT-PCR. The obtained cDNA sequences were used as queries in BLAST P searches against NCBI. The open reading frame (ORF) and amino acid sequence were analyzed by DNAMAN 6.0, and phylogenetic tree was constructed by MEGA 6 software. The conserved domains were predicted by Pfam 26.0 and Conserved Domains in NCBI. CELLO v.2.5, PSORT and SoftBerry ProtComp 9.0 were used for predicting subcellular location. As a result, thirteen MdNAC cDNAs from ‘Zihong Fuji’ were obtained (designated MdNAC40-45, MdNAC47-53, with GenBank accession No. MG099877-MG099882, MG099884-MG099890), with sizes of open reading frame (ORF) of 639 - 1830 bp. By phylogenetic analyses, MdNAC41 was assigned to AtNAC3 group, MdNAC40, MdNAC42-43 and MdNAC45-53 were NAM group, and MdNAC44 was belonged to VND group. The subcellular localization prediction suggested that MdNAC40-45, MdNAC47-50 and MdNAC52-53 were in the nucleus, while MdNAC51 was located in the cytoplasm or nucleus. The results from array analysis indicated that thirteen MdNAC genes were expressed in all examined tissues with various transcript abundance. RNA-seq data showed that the expression levels of MdNAC43, MdNAC45, MdNAC47, MdNAC49 and MdNAC51 were induced in response to AAAP infection. qRT-PCR results showed that, under 150 mmol/L NaCl treatment, the transcription levels of MdNAC45, MdNAC47 and MdNAC52 were induced, whereas the transcription levels of MdNAC40, MdNAC48 and MdNAC51 were down-regulated in ‘Gala’ tissue culture seedlings under 300 mmol/L mannitol treatment, the transcription level of MdNAC50 was induced, while the transcription level of MdNAC41 was down-regulated. These results indicated that 13 MdNAC genes were expressed in all examined tissues, and these genes were inducible under AAAP infection, NaCl or mannitol treatment with different degrees. Taken together, these results laid a theoretical foundation to further study the mechanism of NAC transcription factor in the regulation of apple growth, development and stress responses.

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李慧峰,董庆龙,赵强,等.13个苹果NAC转录因子基因的克隆与表达分析[J].植物遗传资源学报,2019,20(4):1041-1051.

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  • 收稿日期:2018-12-05
  • 最后修改日期:2019-05-20
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