留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

瘤背石磺FMRFamide基因的克隆、分析及其在炎症刺激下的表达变化

李称奇 土志涵 吴荣宇 赵枫燊 贾晶晶 沈和定

李称奇, 土志涵, 吴荣宇, 赵枫燊, 贾晶晶, 沈和定. 瘤背石磺FMRFamide基因的克隆、分析及其在炎症刺激下的表达变化[J]. 南方水产科学, 2021, 17(2): 70-77. doi: 10.12131/20200167
引用本文: 李称奇, 土志涵, 吴荣宇, 赵枫燊, 贾晶晶, 沈和定. 瘤背石磺FMRFamide基因的克隆、分析及其在炎症刺激下的表达变化[J]. 南方水产科学, 2021, 17(2): 70-77. doi: 10.12131/20200167
Chengqi LI, Zhihan TU, Rongyu WU, Fengshen ZHAO, Jingjing JIA, Heding SHENG. Cloning and expression analysis of FMRFamide gene in Onchidium reevesii and inflammatory stimulaton on its gene expression[J]. South China Fisheries Science, 2021, 17(2): 70-77. doi: 10.12131/20200167
Citation: Chengqi LI, Zhihan TU, Rongyu WU, Fengshen ZHAO, Jingjing JIA, Heding SHENG. Cloning and expression analysis of FMRFamide gene in Onchidium reevesii and inflammatory stimulaton on its gene expression[J]. South China Fisheries Science, 2021, 17(2): 70-77. doi: 10.12131/20200167

瘤背石磺FMRFamide基因的克隆、分析及其在炎症刺激下的表达变化

doi: 10.12131/20200167
基金项目: 国家自然科学基金项目 (41276157);上海高校水产学一流学科建设项目
详细信息
    作者简介:

    李称奇 (1995—),男,硕士研究生,研究方向为海洋贝类免疫学。E-mail: helieo1@163.com

    通讯作者:

    沈和定 (1964—),男,博士,教授,从事海洋贝类遗传育种研究。E-mail: hdshen@shou.edu.cn

  • 中图分类号: S 917.4

Cloning and expression analysis of FMRFamide gene in Onchidium reevesii and inflammatory stimulaton on its gene expression

  • 摘要: 该文研究了瘤背石磺 (Onchidium reevesii) 在炎症刺激后体内FMRFamide基因的表达水平变化,以及FMRFamide多肽保持其自身稳态的分子机制。以瘤背石磺转录组中FMRFamide基因片段为基础,通过RACE (Rapid-amplification of cDNA ends) 技术克隆得到该基因cDNA的全长为2 618 bp,开放阅读框 (Open reading frame, ORF) 为882 bp,编码293个氨基酸,系统进化树显示该基因与静水椎实螺 (Lymnaea stagnalis) FMRFamide基因的亲缘关系最近,这和传统形态学分类相吻合。实时荧光定量结果显示FMRFamide基因在瘤背石磺的不同组织中均有表达,但在神经节部位的相对表达量极显著高于皮肤、腹足、肝胰腺、血细胞、性腺和肌肉组织 (P<0.01)。免疫组织化学验证了FMRFamide多肽在组织中与mRNA分布的一致性。炎症刺激实验结果表明,注射脂多糖 (LPS) 后,实验组神经节、肝胰腺、血细胞和皮肤中FMRFamide基因的mRNA表达水平显著高于对照组 (P<0.05),且在刺激后第12小时相对表达量达到最高。综上,瘤背石磺FMRFamide主要存在于神经节中,通过神经内分泌系统参与肌体的免疫调节,对炎症刺激下的瘤背石磺保持体内稳态具有重大作用。
  • 图  1  FMRFamide基因的氨基酸序列及cDNA全长

    浅灰色阴影部分是该基因编码信号肽部分,起始密码子和终止密码子用方框标出,深灰色阴影是磷酸化位点,末端加尾信号AATAAA用下划线标出,poly (A) 用斜体表示

    Figure  1.  Amino acid sequence and full length cDNA of FMRFamide gene

    The light grey shaded portion is the signal peptide portion of the gene; the start and stop codons are boxed; the dark grey shaded portion is the phosphorylation site; the end plus tail signal AATAAA is underlined, and poly (A) is in italics.

    图  2  利用MEGA-X软件基于NJ法构建的FMRFamide系统进化树

    Figure  2.  NJ phylogenetic tree of FMRFamide by MEGA-X

    图  3  OrFMRFamide在各组织的相对表达量

    **. 极显著性差异 (P<0.01)

    Figure  3.  mRNA relative expression of OrFMRFamide in different tissues

    **. Very significant at 0.01 level (P<0.01)

    图  4  免疫组化结果

    a. 神经节;b. 腹足;c. 肝胰腺;d. 肌肉;e. 皮肤;f. 性腺;箭头表示FMRFamide多肽

    Figure  4.  Immunohistochemistry results

    a. Ganglia; b. Pleopod; c. Hepatopancreas; d. Muscle; e. Skin; f. Gonad; arrows represent FMRFamide polypeptides.

    图  5  OrFMRFamide基因在炎症刺激后在不同组织中的相对表达量

    图中不同字母表示差异显著 (P<0.05)

    Figure  5.  Relative expression of OrFMRFamide gene in different tissues after inflammatory stimulation

    Different letters in the figure indicate significant difference (P<0.05).

    表  1  实验中所用引物序列

    Table  1.   Primers used in this experiment

    引物
    Primer
    引物序列 (5'–3')
    Primer sequence
    用途
    Function
    OrFMRFamide-F1 CTTAGGAGTGGGAACAGC 验证目的片段
    OrFMRFamide-R1 CGGCTGGAGATTTGATTG
    OrFMRFamide-F2 CGGACCAGTACGACCAAC
    OrFMRFamide-R2 GTTCAGTCCGCCCTAATG
    OrFMRFamide-3'RACE-inter ACTGGTTTGGGTAGCA 3'RACE 特异性引物
    OrFMRFamide-3'RACE-outer ATGGCAACAATGTCTTTCG
    OrFMRFamide-5'RACE-inter GAGCAGAAGATGGCGT 5'RACE 特异性引物
    OrFMRFamide-5'RACE-outer TCCTCGCTTTGCCTCA
    OrFMRFamide-RT-F AGCTGGAGGACACACTGAGGCA Real-time RT-PCR
    OrFMRFamide-RT-R TGCCACATCGCCCTCATCGG
    18S-F TCCGCAGGAGTTGCTTCGAT
    18S-R ATTAAGCCGCAGGCTCCACT
    下载: 导出CSV
  • [1] 蔺洋春, 崔光旭, 王晓安. 神经肽Myomodulin研究进展[J]. 鲁东大学学报 (自然科学版), 2016, 32(2): 143-151.
    [2] PRICE D A, GREENBERG M J. The structure of a molluscan cardioexcitatory neuropeptide FMRFamide[J]. Science, 1977, 197: 670-671. doi: 10.1126/science.877582
    [3] 潘恒倩. 脉红螺 (Rapana venosa) 胚胎和幼虫神经系统发育的初步研究[D]. 青岛: 中国海洋大学, 2015: 1-63.
    [4] MARCINIAK P, WITEK W, SZYMCZAK M, et al. FMRFamide-related peptides signaling is involved in the regulation of muscle contractions in two tenebrionid beetles[J]. FPHYS, 2020, 5(11): 456-469.
    [5] KIM M A, MARKKANDAN K, HAN N Y, et al. Neural ganglia transcriptome and peptidome associated with sexual maturation in female Pacific abalone (Haliotis discus hannai)[J]. Genes, 2019, 10(4): 1-13.
    [6] ROSZER T, KAPPELMAYER J, NAGY G G, et al. The neuropeptide FMRFamide can protect cells against apoptosis in the snail digestive gland[J]. Apoptosis, 2006, 11(2): 173-182. doi: 10.1007/s10495-006-3391-4
    [7] LI M J, WANG M, WANG W L, et al. The immunomodulatory function of invertebrate specific neuropeptide FMRFamide in oyster Crassostrea gigas[J]. Fish Shellfish Immunol, 2019, 88: 480-488. doi: 10.1016/j.fsi.2019.03.023
    [8] GUAN Z H, KIM J H, LOMVARDS S, et al. P38 MAP kinase mediates both short-term and long-term synapticdepression in aplysia[J]. J Neurosci, 2003, 23(19): 7317-7325. doi: 10.1523/JNEUROSCI.23-19-07317.2003
    [9] 段晓伟. 瘤背石磺 (Onchidium struma) 的生物学研究[D]. 上海: 华东师范大学, 2007: 1-90.
    [10] SHEN H, LI K, CHEN H, et al. Experimental ecology and hibernation of Onchidium struma (Gastropoda: Pulmonata: Systellommatophora)[J]. J Exp Mar Biol Ecol, 2011, 396(2): 71-76. doi: 10.1016/j.jembe.2010.09.010
    [11] SHEN H, WANG L, DAI X, et al. Karyotypes in Onchidium struma (Gastropoda: Pulmonata: Systellommatophora)[J]. Moll Res, 2010, 30(2): 113-116.
    [12] NA Z, SHEN H, CHENG C, et al. Genetic structure of Onchidium] "struma" (Mollusca: Gastropoda: Eupulmonata) from the coastal area of China based on mtCO I[J]. Mit DNA A, 2016, 27(2): 1319-1323.
    [13] 张坤霞. 我国石磺科形态结构再描述及同工酶分析[D]. 上海: 上海海洋大学, 2013: 1-67.
    [14] BINGNING G, WEI L, TIEZHU Y, et al. Metallothionein, hemocyte status and superoxide dismutase/aspartate aminotransferase activity are sensitive biomarkers of cadmium stress in Onchidium reevesii[J]. Aquat Toxicol, 2019, 215: 105284. doi: 10.1016/j.aquatox.2019.105284
    [15] VERBURG-VAN K B M L, VAND A L M, CHADZINSKA M. Neuroendocrine-immune interaction: regulation of inflammation via G-protein coupled receptors[J]. Gen Comp Endocrinol, 2013, 188: 94-101. doi: 10.1016/j.ygcen.2012.11.010
    [16] ESTURDO L V, AGUILAR M B, COTERA E P H D L. FMRFamide and related peptides in the phylum mollusca[J]. Peptides, 2008, 29(2): 310-317. doi: 10.1016/j.peptides.2007.09.025
    [17] 杨金龙, 李树恒, 刘志伟, 等. 厚壳贻贝胚胎和早期幼虫神经系统发育的初步研究[J]. 水产学报, 2013(4): 512-519.
    [18] KIM M A, KESAVAN M, HAN N Y, et al. Neural ganglia transcriptome and peptidome associated with sexual maturation in female Pacific abalone (Haliotis discus hannai)[J]. Genes, 2019, 268(10): 1-21.
    [19] YUKO M, AMAMI Y, RYOTA M. RFamidergic neurons in the olfactory centers of the terrestrial slug Limax[J]. Zool Lett, 2018, 4(22): 1-15.
    [20] ANNA D C, CARLO D C. Neuropeptidergic control of the optic gland of Octopus vulgaris: FMRF-amide and GnRH immunoreactivity[J]. J Comp Neurol, 2015, 398(1): 1-12.
    [21] 高凤娟, 李丕鹏, 陆宇燕. 花背蟾蜍皮肤结构及其抗氧化和免疫功能的研究[J]. 沈阳师范大学学报 (自然科学版), 2015(3): 324-328.
    [22] ALLAN M, BOYLE M J, RICE M E, et al. Developmental architecture of the nervous system in Themiste lageniformis (Sipuncula): new evidence from confocal laser scanning microscopy and gene expression[J]. J Morphol, 2019, 280: 1628-2650. doi: 10.1002/jmor.21054
    [23] TERENINA N B, KRESHCHENKO N D, MOCHALOVA N V, et al. The new data on the serotonin and FMRFamide localization in the nervous system of Opisthorchis felineus metacercaria[J]. Acta Parasitol, 2020, 65(2): 361-374. doi: 10.2478/s11686-019-00165-2
    [24] WANG W, LI M, WANG L, et al. The granulocytes are the main immunocompetent hemocytes in Crassostrea gigas[J]. Dev Comp Immunol, 2017, 2(67): 221-228.
    [25] ZHAOQUN L, LINGLING W, ZHAO L, et al. The cholinergic and adrenergic autocrine signaling pathway mediates immunomodulation in oyster Crassostrea gigas[J]. Front Immunol, 2018, 9: 1-10. doi: 10.3389/fimmu.2018.00001
    [26] MCGUIRE S R, LIST R P, DAY D E, et al. Phagocyte-derived catecholamines enhance acute inflammatory injury[J]. Nature, 2008, 449(7163): 721-725.
    [27] MALAGOLI D, MANDRIOLI M, TASCEDDA F, et al. Circulating phagocytes: the ancient and conserved interface between immune and neuroendocrine function[J]. Bio Rev, 2017, 92(1): 195-201.
    [28] WANG T, MIN Z, DI L, et al. Changes in the neuropeptide content of Biomphalaria ganglia nervous system following Schistosoma infection[J]. Parasit Vectors, 2017, 10: 1-13. doi: 10.1186/s13071-016-1943-1
    [29] HADA A, KUMARI C, PHANI V, et al. Host-induced silencing of FMRFamide-like peptide genes, flp-1 and flp-12, in rice impairs reproductive fitness of the root-knot nematode Meloidogyne graminicola[J]. Front Plant Sci, 2020, 11: 1-10. doi: 10.3389/fpls.2020.00001
    [30] KERBL A, TOLSTRUP E W, WORSAAE K. Nerves innervating copulatory organs show common FMRFamide, FVRIamide, MIP and serotonin immunoreactivity patterns across Dinophilidae (Annelida) indicating their conserved role in copulatory behaviour[J]. BMC Zool, 2019, 8(4): 1-13.
  • 加载中
图(5) / 表(1)
计量
  • 文章访问数:  87
  • HTML全文浏览量:  44
  • PDF下载量:  8
  • 被引次数: 0
出版历程
  • 收稿日期:  2020-08-13
  • 修回日期:  2020-11-17
  • 网络出版日期:  2021-02-04
  • 刊出日期:  2021-04-05

目录

    /

    返回文章
    返回