Identification and Functional Characterization of Ammonia transporter Genes PdRhp-1 in Pocillopora damicornis
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摘要: 为揭示高温对鹿角杯形珊瑚（Pocillopora damicornis）氨同化作用的影响，阐明造礁石珊瑚的热适应机制，本研究鉴定并克隆了1个鹿角杯形珊瑚氨转运体基因 PdRhp-1，其开放阅读框（ORF）全长
1410 bp，编码一条由469 aa组成的多肽链。序列分析结果表明，PdRhp-1 是一种疏水性跨膜蛋白，含有12个跨膜结构域，属于 Rhesus 型氨转运体，其氨基酸序列与人（Homo sapiens）的氨转运体基因 RhCG 的一致性为44.14%。为解析 PdRhp-1 的生物学功能，将其重组表达载体转染至 HEK293T 细胞，并在培养基中添加氯化铵，发现表达 PdRhp-1 的细胞总氨摄取率显著高于对照组，说明 PdRhp-1 具有氨转运功能。同时，分析了高温处理鹿角杯形珊瑚的转录组数据，发现高温显著抑制了 PdRhp-1 及部分氨同化相关基因的表达。以上结果确认，鹿角杯形珊瑚 PdRhp-1 是一个 Rhesus 型氨转运体，高温可能通过抑制 PdRhp-1 介导的氨转运过程影响珊瑚与虫黄藻的共生稳态。Abstract: To reveal the effect of high temperature on the ammonia assimilation of Pocillopora damicornis and elucidate the thermal adaptation mechanism of scleractinian coral, we identified and cloned an ammonia transporter gene PdRhp-1 from P. damicornis. The open reading frame (ORF) of PdRhp-1 is1410 bp, encoding a polypeptide chain composed of 469 amino acid residues. Sequence analysis showed that PdRhp-1 was a hydrophobic transmembrane protein, containing 12 transmembrane domains, belonging to the Rhesus-type ammonia transporter. Its amino acid sequence shares 44.14% identity with Homo sapiens ammonia transporter gene RhCG. In order to analyze the biological function of PdRhp-1, its recombinant expression vector was transfected into HEK293T cells, and ammonium chloride was added to the culture medium. It was found that the total ammonia uptake rate in the cells expressing PdRhp-1 was significantly higher than that in the control group, indicating that PdRhp-1 has ammonia transport function. At the same time, the transcriptome data of high temperature treated P. damicornis were analyzed, and it was found that high temperature significantly inhibited the expression of PdRhp-1 and some genes related to ammonia assimilation. The above results demonstrate that PdRhp-1 is a Rhesus-type ammonia transporter, and high temperature may affect the symbiotic homeostasis of corals and zooxanthellae by inhibiting the ammonia transport process mediated by PdRhp-1.-
Key words:
- Pocillopora damicornis /
- Ammonia transporter /
- Ammonia assimilation /
- High temperature
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图 1 PdRhp-1 的 Ammonium-transp 结构域（A）、跨膜区（B）、二级结构（C）和三级结构（D）
Fig. 1 The Ammonium-transp domain (A), transmembrane region (B), secondary structure (C) and tertiary structure (D) of PdRhp-1
图 2 PdRhp1 与其他生物氨转运体共同构建的最大似然树
Rh 家族聚为5支，分别对应Rh30（橙）、RhAG（紫）、RhBG（蓝）、RhCG（绿）、Rhp1（粉）等亚型。AMT 家族聚为一支（棕）。
Fig. 2 The maximum likelihood tree constructed by PdRhp1 and other biological ammonia transporters
The Rh family is divided into five subfamilies, corresponding to the subtypes Rh30(orange), RhAG(purple), RhBG(blue), RhCG(green), Rhp1(pink), and AMT family clustered into one branch (brown).
图 3 鹿角杯形珊瑚 PdRhp-1 与人 RhCG 的多序列比对
N-糖基化位点（棕色Δ）；苯丙氨酸门（蓝色Δ）；胞质分流残基：保守（绿色）和不匹配（粉色）；双组氨酸（红色☆）；天冬氨酸残基（黑色○）；疏水通道内壁残基：保守（浅蓝）和不匹配（橙色）；间隙用黑点表示
Fig. 3 Multiple Sequence Alignment of P. damicornis PdRhp-1 and Homo sapiens RhCG
N-glycosylation (brown Δ); Phenylalanine gate (blue Δ); Cytoplasmic shunt residues: conserved (green) and mismatched (pink); double histidine residues (red ☆); aspartic acid residues (black ○); hydrophobic channel lining residues: conserved (light blue) and mismatched (orange); gaps marked with black dots
图 4 pEGFP-N1-PdRhp-1 重组质粒图谱（A）和平均 Tamm 摄取率（B）
平均 Tamm 摄取率表示每6.25×106个细胞/毫升1 min的 Tamm 摄取率；*表示存在显著差异，p < 0.05
Fig. 4 Recombinant plasmid map of pEGFP-N1- PdRhp-1(A) and average Tamm uptake rate(B)
The average Tamm uptake rate was expressed as the Tamm uptake rate per 6.25 × 106 cells/mL for 1 min; * means there is a significant difference, p < 0.05
图 5 氨同化相关基因在热胁迫过程中的差异基因表达热图
S0：初始状态；S1：水螅体收缩；S2：共肉分离；S3：水螅体完全脱离
Fig. 5 Differential gene expression heat map of ammonia assimilation related genes in the process of heat stress
S0: initial stage; S1: polyp contraction; S2: coenosarc separation; S3: polyp detachment
表 1 本研究使用到的主要引物及信息
Tab. 1 Primer sequence and related information
引物名称 序列（5’-3’） 引物用途 PdEF-CerF CAGGATGTTTACAAGATTGGAGGT 内参引物 PdEF-CerR TTGTCACTTTGCCAGCGACTT 内参引物 F1 TAAGTCGCACCCCTCTATCAGTC 基因克隆 R1 CCCAAAATCCCTTCTTGCTTAT 基因克隆 F2 ATGGGCTTCAAATTTTCTCTCATCAG 基因克隆 R2 TTAAACTTTAGATACTGCTTCCGCTT 基因克隆 HindⅢ-F CCCAAGCTTGCCACCATGGGCTTCAAATTTTCTCTCATCAGTG 重组表达载体构建 BamHI-R CGCGGATCCGCTTTAGATACTGCTTCCGCTTCGCC 重组表达载体构建 M13-47 CGCCAGGGTTTTCCCAGTCACGAC pMD19-T 载体引物 RV-M AGCGGATAACAATTTCACACAGGA pMD19-T 载体引物 pEGFP-N5 CGGTGGGAGGTCTATATAAG pEGFP-N1 载体引物 pEGFP-N3 GTCGCCGTCCAGCTCGACCAG pEGFP-N1 载体引物 
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