克氏原螯虾受鲶爱德华氏菌刺激后<i>Pc-crustin</i> 6基因的表达分析

杨兵兵; 魏哲; 蔺思函; 李倩倩; 李博; 王春丹; 沈秀丽; 杜志强

doi:10.19303/j.issn.1008-0384.2021.09.008

克氏原螯虾受鲶爱德华氏菌刺激后Pc-crustin 6基因的表达分析

doi: 10.19303/j.issn.1008-0384.2021.09.008

1.
内蒙古科技大学生命科学与技术学院，内蒙古自治区　包头　014010
2.
内蒙古科技大学图书馆，内蒙古自治区　包头　014010

基金项目: 内蒙古自治区自然科学基金（2020JQ03）；国家自然科学基金（32060834、31660260）

详细信息

作者简介:
杨兵兵（1994−），男，硕士研究生，研究方向：无脊椎动物的先天免疫（E-mail：2633159278@qq.com）

通讯作者:
杜志强（1980−），男，博士，教授，研究方向：无脊椎动物的先天免疫（E-mail：nmdzq1981@163.com）

中图分类号: Q 786
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- 被引次数: 0
出版历程
- 收稿日期: 2021-04-12
- 修回日期: 2021-06-24
- 网络出版日期: 2021-08-10
- 刊出日期: 2021-09-28

Gene Cloning and Expression of Crustin 6 in Procambarus clarkii

1.
College of Life Science and Technology, Inner Mongolia University of Science and Technology, Baotou, Inner Mongolia　014010, China
2.
Library, Inner Mongolia University of Science and Technology, Baotou, Inner Mongolia　014010, China

摘要

摘要: 目的为了后续深入研究克氏原螯虾甲壳肽基因的功能。方法以Pc-crustin 6甲壳肽基因作为研究对象，通过设计特异性引物进行PCR扩增，利用生物信息学软件进行氨基酸序列比对与系统进化树分析，探讨Pc-Crustin 6的分子特性；并利用qRT-PCR技术分析目的基因在不同组织中的表达水平，检测鲶爱德华氏菌刺激后基因的表达模式。结果 Pc-crustin 6的cDNA序列全长为465 bp，开放阅读框包括384 bp，编码127个氨基酸残基；在分子的N端包含一个由25个氨基酸残基组成的信号肽序列，C端包含一个由8个保守的半胱氨酸残基构成的WAP结构域。Pc-crustin 6在被检测的5个组织中均存在一定的表达，其中，鳃组织的相对表达量最高。鲶爱德华氏菌刺激后，Pc-crustin 6在血细胞、肝胰腺、鳃、肠组织中均表现出了明显的上调趋势。结论机体在受到鲶爱德华氏菌刺激后，Pc-crustin 6相对表达量发生改变，推测其在小龙虾抗鲶爱德华氏菌过程中发挥了作用。
- 克氏原螯虾 /
- 抗菌肽 /
- 基因克隆 /
- 先天免疫 /
- 生物信息学分析
Abstract: Objective Functions of crustacean gene of Procambarus clarkia were studied. Method Pc-crustin6 was targeted for this study with specifically designed primers for PCR. Amino acids alignment and phylogenetic tree analysis were performed with bioinformatics software to decipher the molecular properties of Pc-crustin 6. Expressions and distributions of Pc-crustin 6 in different tissues were analyzed by qRT-PCR. The expression after an Edwardsiella ictaluri challenge was used to determine the disease resistance associated with the gene. Result The full length Pc-crustin 6 cDNA was 465 bp with an open reading frame of 384 bp and encoding 127 amino acids. The N terminal of the molecule contained a signal peptide of 25 amino acids, and the C terminal a WAP domain of 8 conserved cysteine residues. Pc-crustin 6 was expressed to varied extents in 5 organs including the gills, which was the highest among them. After an artificial exposure to E. ictaluri, the expressions of Pc-crustin 6 in the hemocytes, hepatopancreas, gills, and intestines of P. clarkia were significantly up-regulated. Conclusion The Pc-crustin 6 expression in organs of P. clarkia was significantly altered after being stimulated by the induction of E. ictaluri. It indicated that the gene was involved in the pathogenic resistance of P. clarkia.
- Procambarus clarkii /
- antimicrobial peptides /
- gene cloning /
- innate immunity /
- bioinformatic analysis

HTML全文

图 1 Pc-crustin 6基因PCR扩增后的电泳结果

注：M：标准DNA分子量；1～3：目的基因的PCR扩增条带。

Figure 1. Electrophoresis of Pc-crustin 6 after PCR amplification

Note: M: standard DNA marker; 1-3: target gene PCR amplification bands.

下载: 全尺寸图片幻灯片

图 2 Pc-crustin 6的cDNA序列以及编码的氨基酸序列

注：下划线为信号肽序列，灰色底纹区域为WAP结构域，右侧数字分别代表核苷酸与氨基酸残基的数目。

Figure 2. cDNA and encoded amino acids sequences of Pc-crustin 6

Note: Signal peptide sequence is underlined; grey area represents WAP domain; numbers on the right represent nucleotide and corresponding amino acids sequences.

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图 3 Pc-crustin 6与其他物种crustin基因的氨基酸序列比对结果

Figure 3. Amino acids sequence alignment between Pc-crustin 6 and other crustins

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图 4 Pc-Crustin 6分子与其他Crustin分子亲缘关系的进化树分析

Figure 4. Phylogenetic analysis on relationships between Pc-Crustin 6 and other Crustins

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图 5 Pc-crustin 6基因的多组织表达谱分析

Figure 5. Expressions of Pc-crustin 6 in various tissues of P. clarkia

下载: 全尺寸图片幻灯片

图 6 鲶爱德华氏菌刺激后Pc-crustin 6的表达模式

注：①鲶爱德华氏菌刺激后Pc-crustin 6在四种组织中的相对表达模式，A血细胞的相对表达模式，B肝胰腺的相对表达模式，C鳃组织的相对表达模式，D肠组织的相对表达模式.②*表示显著差异（P<0.05），**表示极显著差异（P<0.01）。

Figure 6. Pc-crustin 6 expressions after E. ictaluri stimulation

Note: ①Relative expressions of Pc-Crustin 6 in tissues of P. clarkia after Edwardsiella cataphylosis; A: blood cells; B: hepatopancreas; C: gills; D: intestines.②* represents significant difference (P<0.05), ** represents extremely significant difference (P<0.01).

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