|
|
Identification, Functional Analysis of the Rh Gene Family in Sinonovacula constricta and Screening of It's SNPs Associated with Ammonia Nitrogen Tolerance |
LAI Cong-Ying1,2, LIU Zi-Dai1,2, DONG Ying-Hui1,3, LYU Li-Yuan1,2,*, LIN Zhi-Hua1,* |
1 Zhejiang Key Laboratory of Aquatic Germplasm Resources/College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China; 2 Ninghai Institute of Mariculture Breeding and Seed Industry, Zhejiang Wanli University, Ninghai 315604, China; 3 College of Advanced Agricultural Sciences, Zhejiang Wanli University, Ningbo 315101, China |
|
|
Abstract Rhesus proteins (Rh) belong to the ammonia transporter gene family and play an important role in the ammonia excretion of animals. Sinonovacula constricta, as a main economic shellfish in mixed culture in confined ponds, often faces severe high ammonia nitrogen environment due to its buried lifestyle. In this study, the gene family of Rh transporters in S. constricta (Sco-Rhs) was identified at the genome-wide level by bioinformatics methods. Subsequently, their expression patterns under ammonia stress, gene functions, and correlation with ammonia-nitrogen tolerance were investigated. The results showed that 2 Sco-Rhs genes (Sco-Rhbg-1和Sco-Rhbg-2) were identifed from the S. constricta genome. Moreover, the homology between Sco-Rhs was 57.96%. The tissue expression results showed that Sco-Rhs genes expressed in all tissues, with the highly specific expression in gills (P<0.01). And Sco-Rhs had different expression patterns under ammonia stress. With the increase of stress time, the expression of Sco-Rhbg-1 gene in gills showed a downward trend and had a stronger response to ammonia stress. The expression of Sco-Rhbg-2 gene firstly down-regulated and then increased, and finally approached to the initial level. In addition, heterologous yeast complementation was used to study the function of Sco-Rhs. The results showed that the Sco-Rhbg-1 protein is a functional ammonia transporter, and its ability to transport NH4+ was not affected by external pH. And 15 SNPs in the CDS region of Sco-Rhbg-1 were significantly associated with the ammonia tolerance of S. constricta. These results indicated that the Sco-Rhbg-1 gene played an important role in the regulation of ammonia stress in S. constricta. This study provides an important theoretical basis for further exploration of the molecular regulation of Sco-Rhs in high ammonia nitrogen adaptation and molecular marker-assisted breeding.
|
Received: 06 January 2025
|
|
Corresponding Authors:
*llyuan.2009@163.com; zhihua9988@126.com
|
|
|
|
[1] 丛郁, 杨顺瑛, 金曼, 等. 2013. 豆梨铵转运蛋白基因PcAMT1-1和PcAMT1-2的克隆与功能鉴[J]. 园艺学报, 40(11): 2115-2126. (Cong Y, Yang S Y, Jin M, et al.2013. Molecular cloning and function analyses of two ammonium transporter protein genes from Pyrus calleryana[J]. Acta Horticulturae Sinica, 40(11): 2115-2126.) [2] 董晓丽, 位莹莹,徐奇友. 2013. 鲤(Cyprinus carpio) Rh糖蛋白家族基因的克隆与组织mRNA表达织mRNA表达[J]. 水产学杂志, 26(5): 6-10. (Dong X L, Wei Y Y, Xu Q Y.2013. Cloning and expression of Rhesus glycoprotein genes in tissues in common carp (Cyprinus carpio)[J]. Chinese Journal of Fisheries, 26(5): 6-10.) [3] 董晓丽, 位莹莹, 徐奇友. 2014. 大豆蛋白源饲料中添加Gln及其前体物对鲤Rh基因表达和血氨含量的影响[J]. 水产学杂志, 27(5): 19-23. (Dong X L, Wei Y Y, Xu Q Y.2014. Effects of glutamine and its precursors on Rh genes expressions and content of blood ammonia of in common carp (Cyprinus carpio)[J]. Chinese Journal of Fisheries, 27(5): 19-23.) [4] 赖卓欣, 宋欣霖, 潘若哲, 等. 2024. 马氏珠母贝V-ATPase-d基因序列特征及其与耐低温性状的关系[J]. 水产学报, 48(9): 099107. (Lai Z X, Song X L, Pan R Z, et al. 2024. Sequence characte-ristics of V-ATPase-d gene in Pinctada fucata martensii and its relationship with low temperature tolerance[J]. Journal of Fisheries of China, 48(9): 099107.) [5] 杨顺瑛, 丛郁, 郝东利, 等. 2015. 利用异源酵母功能互补法研究水稻铵转运体OsAMT1;1功能及调控机制[J]. 江苏农业科学, 43(1): 27-31. (Yang S Y, Cong Y, Hao D L, et al.2015. The function and regulatory mechanism of ammonium transporter OsAMT1;1 in Oryza sativa were studied by heterologous yeast functional complementing method[J]. Jiangsu Agricultural Sciences, 43(1): 27-31.) [6] 张欢. 2019. 缢蛏对氨氮胁迫的生理响应及分子机制研究[D]. 硕士学位论文, 宁波大学, 导师: 林志华, pp. 9-13. (Zhang H, 2019. Studies on physiological response and molecular mechanisms of the razor clam Sinonovacula constricta to ammonia stress[D]. Thesis for M.S., Ningbo University, Suppervisor: Lin Z H, pp. 9-13) [7] 张欢, 董迎辉, 姚韩韩, 等. 2020. 缢蛏(Sinonovacula constricta) GST和HSP90基因克隆及其在氨氮胁迫下的表达特征分析[J]. 海洋学报, 42(4): 66-78. (Zhang H, Dong Y H, Yao H H, et al.2020. Cloning of GST and HSP90 genes of Sinonovacula constricta and analysis of their expression characteristics under ammonia nitrogen stress[J]. Haiyang Xuebao, 42(4): 66-78.) [8] Andrade S L A, Einsle O.2007. The Amt/Mep/Rh family of ammonium transport proteins (Review)[J]. Molecular Membrane Biology, 24(5-6): 357-365. [9] Bailey T L, Boden M, Buske F A, et al.2009. MEME SUITE: Tools for motif discovery and searching[J]. Nucleic Acids Research, 37: W202-W208. [10] Botstein D, White R L, Skolnick M, et al.2018. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J]. The American Journal of Human Genetics, 31(2): 6-12. [11] Chen C, Chen H, Zhang Y, et al.2020. TBtools: An integrative toolkit developed for interactive analyses of big biological data[J]. Molecular Plant, 13(8): 1194-1202. [12] Cong M, Wu H, Yang H, et al.2017. Gill damage and neurotoxicity of ammonia nitrogen on the clam Ruditapes philippinarum[J]. Ecotoxicology, 26(3): 459-469. [13] Edwards S L, Arnold J, Blair S D, et al.2015. Ammonia excretion in the atlantic hagfish (Myxine glutinosa) and responses of an Rhc glycoprotein[J]. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 308(9): R769-R778. [14] Hall J A, Yan D.2013. The molecular basis of K+ exclusion by the Escherichia coli emmonium channel AmtB[J]. Journal of Biological Chemistry, 288(20): 14080-14086. [15] Hu C X, Dai W F, Zhu X J, et al.2023. Expression and functional analysis of AMT1 gene responding to high ammonia stress in Razor clam (Sinonovacula Constricta)[J]. Animals, 13(10): 1638. [16] Huang C H, Liu P Z.2001. New insights into the Rh superfamily of genes and proteins in erythroid cells and nonerythroid tissues[J]. Blood Cells, Molecules, and Diseases, 27(1): 90-101. [17] Huang C H, Peng J.2005. Evolutionary conservation and diversification of Rh family genes and proteins[J]. Proceedings of the National Academy of Sciences of the USA, 102(43): 15512-15517. [18] Huang C H, Ye M.2010. The Rh protein family: Gene evolution, membrane biology, and disease association[J]. Cellular and Molecular Life Sciences, 67(8): 1203-1218. [19] Javelle A, Lupo D, Ripoche P, et al.2008. Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB[J]. Proceedings of the National Academy of Sciences of the USA, 105(13): 5040-5045. [20] Javelle A, Lupo D, Zheng L, et al.2006. An unusual twin-his arrangement in the pore of ammonia channels is essential for substrate conductance[J]. Journal of Biological Chemistry, 281(51): 39492-39498. [21] Khademi S, O'Connell J, Remis J, et al.2004. Mechanism of ammonia transport by Amt/MEP/Rh: Structure of AmtB at 1.35 Å[J]. Science, 305(5690): 1587-1594. [22] Liu S, Pan L, Liu M, et al.2014. Effects of ammonia exposure on nitrogen metabolism in gills and hemolymph of the swimming crab Portunus trituberculatus[J]. Aquaculture, 432: 351-359. [23] Liu Z, Chen Y, Mo R, et al.2000. Characterization of human RhCG and mouse Rhcg as novel nonerythroid Rh glycoprotein homologues predominantly expressed in kidney and testis[J]. Journal of Biological Chemistry, 275(33): 25641-25651. [24] Loqué D, Silvia I M, Susana L A, et al.2009. Pore mutations in ammonium transporter AMT1 with increased electrogenic ammonium transport activity[J]. Journal of Biological Chemistry, 284(37): 24988-24995. [25] Ludewig U, von Wirén N, Frommer W B.2002. Uniport of NH4+ by the root hair plasma membrane ammonium transporter LeAMT1;1[J]. Journal of Biological Chemistry, 277(16): 13548-13555. [26] Lv L Y, Ren J F, Zhang H, et al.2022. Transcriptomic analysis of gill and hepatopancreas in razor clam (Sinonovacula constricta) exposed to acute ammonia[J]. Frontiers in Marine Science, 9: 832494. [27] Lv L Y, Hu C X, Xu H Q, et al.2023. Insight into the genetic basis of ammonia tolerance in razor clam Sinonovacula constricta by genome-wide association study[J]. Aquaculture, 569: 739351. [28] Martin M, Fehsenfeld S, Sourial M M, et al.2011. Effects of high environmental ammonia on branchial ammonia excretion rates and tissue Rh-protein mRNA expression levels in seawater acclimated dungeness crab Metacarcinus magister[J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 160(2): 267-277. [29] McDonald T R, Ward J M.2016. Evolution of electrogenic ammonium transporters (AMTs)[J]. Frontiers in Plant Science, 7: 352. [30] Miranda-Filho K C, Pinho G L L, Wasielesky W, et al.2009. Long-term ammonia toxicity to the pink-shrimp Farfantepenaeus paulensis[J]. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 150(3): 377-382. [31] Nakada T, Westhoff C M, Kato A, et al.2007. Ammonia secretion from fish gill depends on a set of Rh glycoproteins[J]. The FASEB Journal, 21(4): 1067-1074. [32] Nawata C M, Hung C C Y, Tsui T K N, et al.2007. Ammonia excretion in rainbow trout (Oncorhynchus mykiss): Evidence for Rh glycoprotein and H+-ATPase involvement[J]. Physiological Genomics, 31(3): 463-474. [33] Ni Q, Liang X, Yang S, et al.2024. Molecular and physiological responses in the ammonia transport pathways in clam Cyclina sinensis exposed to chronic ammonia nitrogen[J]. Aquaculture Reports, 35: 101952. [34] Ning X, Feng L, Li X, et al.2018. The scallop IGF2 mRNA-binding protein gene PyIMP and association of a synonymous mutation with growth traits[J]. Genes & Genetic Systems, 93(3): 91-100. [35] Peng J, Huang C H.2006. Rh proteins vs amt proteins: An organismal and phylogenetic perspective on CO2 and NH3 gas channels[J]. Transfusion Clinique et Biologique, 13(1-2): 85-94. [36] Perry S F, Braun M H, Noland M, et al.2010. Do zebrafish Rh proteins act as dual ammonia-CO2 channels?[J] Journal of Experimental Zoology Part A: Ecological Genetics and Physiology, 313A(9): 618-621. [37] Pitts R J, Derryberry S L, Pulous F E, et al.2014. Antennal-expressed ammonium transporters in the malaria vector mosquito Anopheles gambiae[J]. PLOS ONE, 9(10): e111858. [38] Quijada-Rodriguez A R, Fehsenfeld S, Marini A-M, et al.2024. Branchial CO2 and ammonia excretion in crustaceans: Involvement of an apical Rhesus-like glycoprotein[J]. Acta Physiologica, e14078. [39] Syvanen A C.2001. Accessing genetic variation: Genotyping single nucleotide polymorphisms[J]. Nature Reviews Genetics, 2(12): 930-942. [40] Tamura K, Stecher G, Kumar S.2021. MEGA11: Molecular evolutionary genetics analysis version 11[J]. F.U. Battistuzzi. Molecular Biology and Evolution, 38(7): 3022-3027. [41] Walker V.2014. Ammonia metabolism and hyperammonemic disorders[J]. Advances in Clinical Chemistry, 67: 73-150. [42] Walsh I M, Bowman M A, Soto Santattiaga I F, et al.2020. Synonymous codon substitutions perturb cotranslational protein folding in vivo and impair cell fitness[J]. Computational Biology, 117(7): 3528-3534. [43] Weihrauch D, Morris S, Towle D W.2004. Ammonia excretion in aquatic and terrestrial crabs[J]. Journal of Experimental Biology, 207(26): 4491-4504. [44] Weihrauch D, Wilkie M P, Walsh P J.2009. Ammonia and urea transporters in gills of fish and aquatic crustaceans[J]. Journal of Experimental Biology, 212(17): 2879-2879. [45] Wright P A, Wood C M.2009. A new paradigm for ammonia excretion in aquatic animals: Role of rhesus (Rh) glycoproteins[J]. Journal of Experimental Biology, 212(15): 2303-2312. [46] Zhang H, Sun G G, Lin Z H, et al.2020. The razor clam Sinonovacula constricta uses the strategy of conversion of toxic ammonia to glutamine in response to high environmental ammonia exposure[J]. Molecular Biology Reports, 47(12): 9579-9593. [47] Zhao X, Fu J, Jiang L, et al.2018. Transcriptome-based identification of the optimal reference genes as internal controls for quantitative RT-PCR in razor clam (Sinonovacula constricta)[J]. Genes & Genomics, 40(6): 603-613. |
|
|
|