柳树NAC基因的克隆与表达模式分析

doi:10.3969/j.issn.1000-2006.201905031

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南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (1): 119-124.doi: 10.3969/j.issn.1000-2006.201905031

柳树NAC基因的克隆与表达模式分析

田雪瑶¹^,²(), 周洁²^,³(), 王保松²^,³, 何开跃¹^,^*(), 何旭东²^,³^,^*()

1.南京林业大学生物与环境学院,江苏南京 210037
2.江苏省林业科学研究院,江苏南京 211153
3.江苏省农业种质资源保护与利用平台柳树资源圃,江苏南京 211153

收稿日期:2019-05-22 修回日期:2019-06-10 出版日期:2020-02-08 发布日期:2020-02-02
通讯作者: 何开跃,何旭东
作者简介:田雪瑶( xyt8389@qq.com),负责实验与数据分析,论文初稿写作;|周洁( 411525402@qq.com),负责实验操作与结果分析。
基金资助:
国家自然科学基金项目(31300556);江苏省林业科学研究院自主科研项目(BM2018022-01)

Cloning and expression pattern analysis of NAC genes in Salix

TIAN Xueyao¹^,²(), ZHOU Jie²^,³(), WANG Baosong²^,³, HE Kaiyue¹^,^*(), HE Xudong²^,³^,^*()

1. College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China
2. Jiangsu Academy of Forestry, Nanjing 211153, China
3. Willow Nursery of the Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing 211153, China

Received:2019-05-22 Revised:2019-06-10 Online:2020-02-08 Published:2020-02-02
Contact: HE Kaiyue,HE Xudong

摘要/Abstract

摘要：

【目的】研究柳树重要抗逆转录因子基因家族,为揭示柳树抗逆分子机制提供理论依据。【方法】以‘苏柳2345’的转录组测序数据为基础,克隆了柳树NAC家族,并分析其序列结构、组织表达特异性以及不同胁迫条件下的表达模式。【结果】克隆的两个柳树NAC转录因子基因分别命名为SlNAC1和SlNAC2。生物信息学分析表明:SlNAC1序列全长为1 126 bp,编码产物含343个氨基酸残基,为稳定的亲水蛋白,定位于细胞核;SlNAC2序列全长为1 139 bp,编码产物含291个氨基酸残基,为稳定的亲水蛋白,定位于叶绿体。两个基因均具有典型的NAM结构域及A、B、C、D、E 5个亚结构域,还包括共同的LPPG、YPNG 和DEE保守基序及NAC抑制结构域。系统进化树分析显示, SlNAC1基因与木薯亲缘关系最近,SlNAC2基因与茄子亲缘关系最近。定量PCR结果显示SlNAC1和SlNAC2均在叶片表达,根中没有表达,为叶片特异性转录因子。定量PCR结果显示SlNAC1基因在脱落酸(ABA)和赤霉素(GA)处理24 h后显著上调,SlNAC2基因在聚乙二醇(PEG)、ABA和GA胁迫后显著上调。【结论】柳树SlNAC1基因在非生物胁迫下表达较为稳定,受ABA和GA胁迫诱导;SlNAC2受干旱、ABA和GA胁迫诱导,受影响明显高于SlNAC1,不受乙烯剂(ETH)胁迫影响。推测SlNAC1和SlNAC2参与GA、ABA信号传导,可能不参与ETH的信号途径。

关键词: 柳树, NAC基因家族, 基因克隆, 基因表达

Abstract:

【Objective】 NAC transcription factors play an important role in regulating plant growth, development and response to stress tolerance. Studying the sequence, structure, evolutionary relationship and expression pattern of NAC transcription factor gene family related with stress tolerance in willow will have important theoretical significance for understanding the molecular mechanism underlying stress tolerance, which in turn will provide a theoretical basis for molecular assisted breeding in future.【Method】 Two members of the NAC gene family were cloned based on the RNA-seq data of the Salix × jiangsuensis ‘J2345’ variety. The gene structure, protein properties and gene phylogeny were analyzed by using bioinformatics. The tissue specific expression in leaf and root, as well as the expression patterns under different types of stress was detected by quantitative real time PCR. 【Result】 The two NAC transcription factors cloned from the leaves of Salix × jiangsuensis ‘J2345’ variety were named SlNAC1 and SlNAC2. Bioinformatics analysis results showed that the sequence lengths of SlNAC1 and SlNAC2 were 1 126 bp and 1 139 bp, encoding proteins with 343 and 291 amino acid residues, respectively. The molecular weight of the proteins expressed from the two SlNAC1 and SlNAC2 genes were 40.1 ku and 42.3 ku, respectively, and both were stable, hydrophilic proteins. The SlNAC1 gene was located in nucleus while the SlNAC2 gene was located in chloroplast. Sequence alignment showed that both the genes contained typical NAM domains as well as A, B, C, D and E sub-domains, which shared the LPPG, YPNG and DEE conservative motifs and NAC suppression domains. Phylogenetic analysis demonstrated that SlNAC1 and SlNAC2 shared the highest homology with the genes from Manihot esculenta and Solanum melongena, respectively. RT-PCR results showed that both SlNAC1 and SlNAC2 were expressed in leaves and not in roots. The results of qRT-PCR showed that SlNAC1 was significantly upregulated after 24 hours of abscisic acid (ABA) and gibberellins (GA) treatments. SlNAC2 was significantly upregulated after exposure to polyethylene glycol (PEG), ABA and GA stresses. 【Conclusion】 The transcription factor SlNAC1 can be induced by induced by ABA and GA, and could stably express under abiotic stress. SlNAC2 can be induced by PEG, ABA and GA rather than ethrel (ETH), at a significantly higher expression level than SlNAC1. Therefore, we speculated that SlNAC1 and SlNAC2 were involved in GA and ABA signal transduction, but not in th e ETH signaling pathway.

Key words: Salix, NAC gene family, gene cloning, gene expression

中图分类号:

S785.5
Q786

田雪瑶,周洁,王保松,等. 柳树NAC基因的克隆与表达模式分析[J]. 南京林业大学学报（自然科学版）, 2020, 44(1): 119-124.

TIAN Xueyao, ZHOU Jie, WANG Baosong, HE Kaiyue, HE Xudong. Cloning and expression pattern analysis of NAC genes in Salix[J].Journal of Nanjing Forestry University (Natural Science Edition), 2020, 44(1): 119-124.DOI: 10.3969/j.issn.1000-2006.201905031.

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PCR类别 PCR type	基因名称 gene name	上下游引物序列 sequences of forward and reverse primers (5'→3')
反转录 reverse transcription	SlNAC1	F:ATGACGGCTACAACACTAGAGTTAC R:TCAAAACGGCTTCTGCAGGTGCATA
反转录 reverse transcription	SlNAC2	F:ATGGGACTGCAAGAAACAGATCCTC R:TCACTGCCTAAACCCAAACCCACTT
实时荧光定量 real time	SlNAC1	F:CCAGCAACTTTCCTGCACAAATATT R:ATGGGACTGCAAGAAACAGATCCTC
实时荧光定量 real time	SlNAC2	F:CGGAGATTGAGGAGGATGAGAAGAA R:GCACGGAGTCTGAGGTGTCAAAGTA

柳树NAC基因的克隆与表达模式分析

Cloning and expression pattern analysis of NAC genes in Salix

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