瘤背石磺<i>FMRFamide</i>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

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

上海海洋大学水产种质资源发掘与利用教育部重点实验室/水产科学国家级实验教学示范中心/海洋动物系统分类与进化上海高校重点实验室，上海 201306

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
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- 文章访问数: 20
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- 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

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

Key Laboratory of Exploration and Utilization of Aquatic Germplasm Resources of Shanghai Ocean University, Ministry of Education/National Experimental Teaching Demonstration Center of Fisheries Science/Shanghai University Key Laboratory of Marine Animal System Classification and Evolution, Shanghai 201306, China

摘要

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) 技术克隆得到了该基因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。
- 瘤背石磺 /
- FMRFamide /
- 基因克隆 /
- 炎症刺激
Abstract: In this study, we investigated the expression of FMRFamide in vivo of Onchidium reevesii after inflammatory stimulus, and the molecular mechanism of FMRFamide polypeptide maintaining homeostasis. Based on a fragment of FMRFamide gene from the transcriptome of O. reevesii, we obtained the full-length cDNA of FMRFamide gene as 2 618 bp by RACE (Rapid-amplification of cDNA ends), including an open reading frame (ORF, 882 bp) which encoded a total of 293 amino acids. The results of the phylogenetic tree suggest that the FMRFamide gene of O. reevesii was most closely related to that of Lymnaea stagnalis, which is consistent with traditional morphological classification. The qRT-PCR results indicate that FMRFamide mRNA was distributed in different tissues of O. reevesii, but the expression in ganglion was very significantly higher than that in skin, hemocyte, hepatopancreas, muscle, gonad and pleopod (P<0.01). Immunohistochemistry verified the consistency of the FMRFamide peptide with mRNA distribution in tissues. The inflammatory stimulation experiment shows that the mRNA expression levels of FMRFamide gene in ganglia, hepatopancreas, hemocyte and skin were significantly higher in the experimental group than in the control group after lipopolysaccharide (LPS) injection (P<0.05), reaching the maximum value at 12^th hour after stimulation. In conclusion, O. reevesii FMRFamide is mainly found in ganglia and is involved in collective immune regulation through neuroendocrine system, playing a significant role in maintaining the stability of tumefaciens in vivo under inflammatory stimuli.
- Onchidium reevesii /
- FMRFamide /
- Genetic cloning /
- Inflammatory stimulus

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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.

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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)

下载: 全尺寸图片幻灯片

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.

下载: 全尺寸图片幻灯片

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性显著 (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).

下载: 全尺寸图片幻灯片

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

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