Cloning and expression analysis of bHLH6 gene from Phyllostachys edulis
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摘要:
目的 研究PhebHLH6转录因子在毛竹Phyllostachys edulis逆境胁迫应答中的作用，为毛竹抗逆分子机制研究奠定一定的基础。 方法 以毛竹实生苗为材料进行非生物胁迫处理[干旱胁迫、盐胁迫、水杨酸(SA)和脱落酸(ABA)处理]，利用转录组数据筛选出1条差异表达基因，命名为PhebHLH6，并对其进行了基因克隆及生物信息学分析；采用实时荧光定量PCR方法分析PhebHLH6在干旱、盐胁迫及SA、ABA处理下的表达模式。 结果 PhebHLH6基因编码区长度为801 bp，编码266个氨基酸，包含bHLH结构域，属于典型的bHLH转录因子。组织特异性表达分析表明：PhebHLH6在毛竹各个组织均有表达，其中在1.5和3.0 m的笋顶部表达丰度最高。在干旱和高盐胁迫处理下，PhebHLH6的表达水平在处理3 h时被强烈诱导，但在处理24 h后显著下调。在SA和ABA激素处理下，PhebHLH6的表达水平被SA和ABA 诱导也呈先上升再下降的趋势，其中受SA强烈诱导，受ABA诱导作用较弱。 结论 PhebHLH6可能参与了毛竹干旱和盐胁迫早期响应途径，并可能在SA和ABA激素信号通路中起一定的调控作用。图4表2参32 Abstract:Objective The objective is to study the role of PhebHLH6 transcription factor in stress response of Phyllostachys edulis; so as to explore the molecular mechanism of resistance to stress in Ph. edulis. Methods Seedlings of Ph. edulis were treated with abiotic stress, including treatments of drought stress, salt stress, salicylic acid (SA) and abscisic acid (ABA). A differentially expressed gene named PhebHLH6 was screened using transcriptome data, and its gene cloning and bioinformatic analysis were performed. Real-time fluorescent quantitative PCR was used to analyze the expression patterns of PhebHLH6 under drought, salt stress and SA and ABA treatments. Result The coding region of PhebHLH6 gene had a base of 801 bp, encoding 266 amino acids, including bHLH domain, which was a typical bHLH transcription factor. Tissue-specific expression analysis showed that PhebHLH6 was expressed in nearly all the tissues of Ph. edulis, with the highest abundance at the top of 1.5 m and 3 m shoots. Under drought and high salt stress, the expression levels of PhebHLH6 were strongly induced after 3 h of treatment but significantly down-regulated after 24 h of treatment. Under SA and ABA hormone treatment, the expression levels of PhebHLH6 increased first and then decreased when induced by SA and ABA, with strong SA induction and weak ABA induction. Conclusion PhebHLH6 may be involved in the early response pathway to drought and salt stress of Ph. edulis and may play a regulatory role in SA and ABA hormone signaling pathways. [Ch, 4 fig. 2 tab. 32 ref.] -
Key words:
- Phyllostachys edulis /
- PhebHLH6 /
- gene clone /
- expression analysis
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图 1 不同物种bHLH6基因氨基酸序列比对
Figure 1 Alignment of bHLH6 amino acid sequences from different species
图 2 不同物种bHLH6-like氨基酸序列进化树构建
Figure 2 Phylogenetic tree construction of BHLH6-like amino acid sequences
图 3 毛竹不同组织中PhebHLH6基因的表达
Figure 3 Expression level of PhebHLH6 gene in different tissues
图 4 非生物胁迫以及激素处理下PhebHLH6表达模式
Figure 4 Expression patterns of PhebHLH6 under abiotic stress and hormone treatment
表 1 基因克隆及表达所用引物序列
Table 1. Primers used in gene clone and quantitative real-time PCR
用途 引物名称 序列(5′→3′) 基因克隆 PhebHLH6-F ATGGACGCGGACATGGGCGACA PhebHLH6-R CTAATAGCTCATCGAGCTCGGG
GGGCTTC实时荧光定量
PCR (RT-qPCR)Q-PhebHLH6-F CGAGAAGCTATACGCGATCC Q-PhebHLH6-F CTGCAGCTGCTGGATGTAAT Q-NTB-F TCTTGTTTGACACCGAAGAGGAG Q-NTB-F AATAGCTGTCCCTGGAGGAGTTT 
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表 2 PhebHLH6基因启动子顺式作用元件分析
Table 2. Cis-element analysis of PhebHLH6 gene promoter
作用元件 序列 数量 功能 作用元件 序列 数量 功能 ABRE CACGTG 9 脱落酸响应元件 Sp1 GGGCGG 1 光响应元件 ARE AAACCA 1 厌氧诱导顺式作用元件 chs-CMA2a TCACTTGA 1 光响应元件 CAAT-box CCAAT 15 启动子和增强子区域调控元件 TGA-element AACGAC 2 生长素响应元件 CGTCA-motif CGTCA 5 茉莉酸甲酯响应元件 O2-site GATGATGTGG 1 玉米醇溶蛋白代谢调节元件 MRE AACCTAA 1 光响应元件 TATA-box TATA 35 核心启动子元件 G-Box TACGTG 3 光响应元件 TGACG-motif TGACG 5 茉莉酸甲酯响应元件
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