澳洲坚果锌指蛋白基因MiZFP9的克隆与功能分析

Cloning and functional analysis of zinc finger protein gene MiZFP9 from macadamia

  • 摘要: 【目的】 克隆澳洲坚果CCCH型锌指蛋白基因MiZFP9,并分析其在不同组织及低温、干旱胁迫下的表达情况,为深入探究澳洲坚果锌指蛋白基因的抗寒和耐旱分子机理提供理论依据。【方法】 从澳洲坚果叶片克隆MiZFP9基因,并对其进行生物信息学分析。通过实时荧光定量PCR检测MiZFP9基因在不同组织中的表达特性及低温和干旱胁迫下的表达模式。【结果】 从澳洲坚果叶片中克隆获得MiZFP9基因,属于CCCH型锌指蛋白基因C3H64亚类,其编码蛋白含有活性位点、Zn2+结合位点及CCCH型锌指蛋白的典型结构域ZF_C3H,为稳定的亲水性蛋白,以丝氨酸磷酸化为主,可能定位于细胞质,属于非分泌型蛋白。MiZFP9蛋白的高级结构主要由无规则卷曲、α-螺旋和延伸链交错组成。MiZFP9基因在Own Choice品种的不同组织中均显著差异表达(P<0.05,下同),其中在刚开放和未开放小花中的相对表达量最高,其次是根和叶,而在茎的相对表达量则最低。在低温胁迫和干旱胁迫下,MiZFP9基因在Own Choice和HAES695品种的相对表达量较对照(处理0 h)均显著上调,其中MiZFP9基因在Own Choice品种中呈先升高后降低的表达模式,在HAES695品种的相对表达量呈波动式变化模式,出现2个峰值,说明其在不同品种中的响应模式存在差异。【结论】 MiZFP9基因具有组织表达特异性,并在低温和干旱处理后表达水平明显升高,推测MiZFP9在澳洲坚果组织生长发育及响应低温和干旱胁迫中发挥重要的正向调控作用。

     

    Abstract: 【Objective】 To clone CCCH type zinc finger protein gene MiZFP9 from macadamia (Macadamia integrifolia) and analyze its expression in different tissues and under cold and drought stresses,which could provide scientific ba‐sis for molecular mechanism of zinc finger protein from macadamia in response to cold and drought stresses.【Method】Gene MiZFP9 was cloned from macadamia leaves,and bioinformatics analysis was conducted.The expression levels of MiZFP9 in different organs of macadamia and under cold and drought stresses were investigated by real-time fluorescence quantitative PCR technology. 【Result】 MiZFP9 gene was cloned from macadamia leaves and belonged to the C3H64 sub‐class of CCCH type zinc finger protein gene.The encoded protein contained active sites,Zn2+ binding sites as well as typi‐cal domains ZF_C3H of CCCH type zinc finger protein,which was a stable hydrophilic protein and was mainly serine phosphorylation.It may be localized in the cytoplasm and was a non-secretory protein.The higher structure of MiZFP9protein was mainly composed of random coil,α-helix and extended chain interlacing.MiZFP9 gene was expressed signifi cantly differently in different tissues of Own Choice variety (P<0.05,the same below),among which the relative expres sion level of MiZFP9 was the highest in newly opened and unopened flowers,followed by roots and leaves,while the relative expression level of MiZFP9 was the lowest in stems.Under low temperature stress and drought stress,the relative expression level of MiZFP9 gene in both Own Choice and HAES695 varieties was significantly up-regulated compared with the control (treated for 0 h),and the expression pattern of MiZFP9 gene was first increased and then decreased in Own Choice variety,the relative expression in HAES695 showed a fluctuating pattern with 2 peaks indicating that the re sponse patters of different varieties were different. 【Conclusion】 MiZFP9 gene is specifically expressed in macadamia tis sues,and is significantly up-regulated under cold and drought stresses,which speculates that MiZFP9 may play importan positive regulatory roles in the growth and development of macadamia tissues and the response to cold and drough stresses.

     

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