蓝莓3个ZIP转运子的克隆及其生物学功能

    Cloning and Biological Function of the Three ZIP Transporters in Blueberry

    • 摘要: 蓝莓Vaccinium spp.的生长代谢极大程度受到金属离子平衡的影响,ZIP转运蛋白家族在调节植物体金属稳态中起着关键性作用,但目前有关蓝莓ZIP转运蛋白的相关研究较少。为探究蓝莓ZIP转运蛋白基因对铁(Fe)、 锌(Zn)、 镉(Cd)等金属离子的吸收转运机制,以北高丛蓝莓布里吉塔为研究材料,对蓝莓ZIP基因家族成员进行克隆,采用生物信息学方法分析得到候选基因的核苷酸序列、氨基酸序列、跨膜结构域,对比其他植物的ZIP家族基因,构建系统进化树并筛选得到高相似性的重点研究对象;通过添加金属水培蓝莓的VcZIPs基因表达量分析,探究蓝莓中ZIP转运子的生物学功能;通过转基因酵母金属耐性分析试验,验证VcZIPs异源表达时的功能。结果表明:克隆得到8个VcZIPs全长cDNA序列,其中筛选出3个转运子VcZIP4、VcZIP7、VcZIP9具有ZIP家族的典型二级结构,或为潜在的ZIP家族成员。q-RT-PCR结果表明,蓝莓叶部ZIP转运子的基因转录水平普遍受到过量金属离子的抑制;蓝莓根部的VcZIPs基因表达普遍受根际过量金属离子诱导,VcZIP4基因的表达量在缺Fe、过量Fe以及超量Cd处理组中分别上调为对照(CK)的5.3、86.5、和45.4倍,VcZIP7基因的表达量在过量Fe处理组中上调为对照的27.2倍,VcZIP 9基因的表达量在过量Cd、超量Cd条件下响应最显著,分别上调为对照(CK)的142.8倍和360.2倍。转基因酵母金属耐性分析试验表明,VcZIP7可能具有镉转运的功能;VcZIP4、VcZIP9均能介导锰的转运而VcZIP7不具备转运锰的能力;VcZIP4、VcZIP7、VcZIP9均具有铁转运功能,其中VcZIP7的铁转运功能最高,其相对较低的表达可能是蓝莓铁营养低效的重要原因。研究结果可为揭示蓝莓ZIP金属转运子家族的运输机制奠定基础。

       

      Abstract: The growth and metabolism of blueberry were greatly affected by the balance of metal ions. ZIP transporter protein family played a key role in regulating the metal homeostasis in plants. However, there were few studies on ZIP transporter protein in blueberry. In order to explore the absorption and transport mechanism of ZIP transporter protein gene in blueberry to the metal ions, such as ferric ion (Fe), zinc (Zn), cadmium (Cd), the members of ZIP gene family in blueberry were cloned by using the northern highbush blueberry Brigitta as the research material. The nucleotide sequence, amino acid sequence and transmembrane domain of the candidate genes were obtained by using the bioinformatics method. Compared with the ZIP family genes of other plants, the phylogenetic tree was constructed and the key research objects with high similarity were screened. The biological function of ZIP transporters in blueberry was explored by analyzing the expression of VcZIPs gene in blueberry with metal hydroponics. The function of heterologous expression of VcZIPs was verified by carrying out the metal tolerance analysis test of transgenic yeast. The results showed that: eight full-length cDNA sequences of VcZIPs were cloned, among which the three transporters, including VcZIP4, VcZIP7 and VcZIP9 had the typical secondary structure of the ZIP family or were the potential members of the ZIP family. The results of qRT-PCR showed that the gene transcription level of ZIP transporter in the leaves of blueberry was generally inhibited by the excessive metal ions. The expression of VcZIPs genes in the roots of blueberry was generally induced by the excessive metal ions in rhizosphere. The expression level of VcZIP4 gene was up-regulated by 5.3, 86.5, and 45.4 times compared with the control (CK) in the deficient Fe, excessive Fe and excessive Cd treatment groups, respectively. The expression level of VcZIP7 gene was up-regulated by 27.2 times compared with the control in the excessive Fe treatment group. The expression level of VcZIP9 gene was the most significant under the conditions of excessive Fe and excessive Cd treatments, which was up-regulated by 142.8 times and 360.2 times compared with the control (CK), respectively. The metal tolerance analysis of transgenic yeast showed that, VcZIP7 might have the function of cadmium transport. Both VcZIP4 and VcZIP9 could mediate the transport of manganese, while VcZIP7 didn't have the ability to transport manganese. VcZIP4, VcZIP7 and VcZIP9 all had the function of iron transport, among which VcZIP7 had the highest function of iron transport, and its relatively low expression might be an important reason for the low efficiency of iron nutrition of blueberry. The results could lay a foundation for revealing the transportation mechanism of the ZIP metal transporter family of blueberry.

       

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