Pomegranate (Punica granatum L.) is an important commercial fruit tree, with moderate tolerance to salinity. The balance of Cl− and other anions in pomegranate tissues are affected by salinity, however, the accumulation patterns of anions are poorly understood. The chloride channel (CLC) gene family is involved in conducting Cl−, NO3−, HCO3− and I−, but its characteristics have not been reported on pomegranate. In this study, we identified seven PgCLC genes, consisting of four antiporters and three channels, based on the presence of the gating glutamate (E) and the proton glutamate (E). Phylogenetic analysis revealed that seven PgCLCs were divided into two clades, with clade I containing the typical conserved regions GxGIPE (I), GKxGPxxH (II) and PxxGxLF (III), whereas clade II not. Multiple sequence alignment revealed that PgCLC-B had a P [proline, Pro] residue in region I, which was suspected to be a NO3−/H+ exchanger, while PgCLC-C1, PgCLC-C2, PgCLC-D and PgCLC-G contained a S [serine, Ser] residue, with a high affinity to Cl−. We determined the content of Cl−, NO3−, H2PO4−, and SO42− in pomegranate tissues after 18 days of salt treatments (0, 100, 200 and 300 mM NaCl). Compared with control, the Cl− content increased sharply in pomegranate tissues. Salinity inhibited the uptake of NO3− and SO42−, but accelerated H2PO4− uptake. The results of real-time reverse transcription PCR (qRT-PCR) revealed that PgCLC genes had tissue-specific expression patterns. The high expression levels of three antiporters PgCLC-C1, PgCLC-C2 and PgCLC-D in leaves might be contributed to sequestrating Cl− into the vacuoles. However, the low expression levels of PgCLCs in roots might be associated with the exclusion of Cl− from root cells. Also, the up-regulated PgCLC-B in leaves indicated that more NO3− was transported into leaves to mitigate the nitrogen deficiency. Our findings suggested that the PgCLC genes played important roles in balancing of Cl− and NO3− in pomegranate tissues under salt stress. This study established a theoretical foundation for the further functional characterization of the CLC genes in pomegranate.

中文翻译：

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所述的全基因组鉴定和表达分析CLC在石榴（基因家族石榴）揭示了盐抗性其角色

石榴（Punica granatum L.）是一种重要的商业果树，对盐分的耐受性中等。石榴组织中Cl-和其他阴离子的平衡受盐度影响，但是，阴离子的积累方式知之甚少。氯离子通道（CLC）基因家族参与了Cl-，NO3-，HCO3-和I-的传导，但石榴的特征尚未见报道。在这项研究中，我们基于门控谷氨酸（E）和质子谷氨酸（E）的存在，鉴定了七个PgCLC基因，由四个反转运蛋白和三个通道组成。系统发育分析表明，七个PgCLC被分为两个进化枝，进化枝I包含典型的保守区GxGIPE（I），GKxGPxxH（II）和PxxGxLF（III），而进化枝II没有。多个序列比对显示，PgCLC-B在区域I中有一个P [脯氨酸，Pro]残基，怀疑是NO3- / H +交换子，而PgCLC-C1，PgCLC-C2，PgCLC-D和PgCLC-G包含在内S [丝氨酸，丝氨酸]残基，对Cl-具有高亲和力。我们在经过18天的盐处理（0、100、200和300 mM NaCl）后，确定了石榴组织中Cl-，NO3-，H2PO4-和SO42-的含量。与对照组相比，石榴组织中的Cl-含量急剧增加。盐度抑制了NO3-和SO42-的吸收，但加速了H2PO4-的吸收。实时逆转录PCR（qRT-PCR）的结果表明，PgCLC基因具有组织特异性表达模式。三种反转运蛋白PgCLC-C1，PgCLC-C2和PgCLC-D在叶片中的高表达水平可能有助于将Cl-螯合到液泡中。然而，根中PgCLC的低表达水平可能与根细胞中Cl-的排除有关。而且，叶片中的PgCLC-B上调表明更多的NO3-被转运到叶片中以减轻氮的缺乏。我们的发现表明，PgCLC基因在盐胁迫下的石榴组织中Cl-和NO3-的平衡中起着重要作用。这项研究为石榴中CLC基因的进一步功能表征奠定了理论基础。我们的发现表明，PgCLC基因在盐胁迫下的石榴组织中Cl-和NO3-的平衡中起着重要作用。这项研究为石榴中CLC基因的进一步功能表征奠定了理论基础。我们的发现表明，PgCLC基因在盐胁迫下的石榴组织中Cl-和NO3-的平衡中起着重要作用。这项研究为石榴中CLC基因的进一步功能表征奠定了理论基础。