| 罗氏沼虾淀粉酶基因克隆及其表达规律分析 |
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| 引用本文： | 姜建萍,杨学明,袁翔,邱庆庆,杨秀荣,蒋钦杨,黄光华,蒋和生.罗氏沼虾淀粉酶基因克隆及其表达规律分析[J].南方农业学报,2021,52(5):1362-1369. |
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| 作者姓名： | 姜建萍 杨学明 袁翔 邱庆庆 杨秀荣 蒋钦杨 黄光华 蒋和生 |
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| 作者单位： | 1. 广西药用植物园, 南宁 530023;2. 广西大学动物科学技术学院, 南宁 530004;3. 广西水产科学研究院, 南宁 530021 |
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| 基金项目： | 广西自然科学基金项目（2018GXNSFBA281209）；广西科技重大专项（桂科AA17204080-6）；广西博士后“两站”项目（T3340097968） |
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| 摘 要： | 【目的】克隆罗氏沼虾（Macrobrachium rosenbergii）淀粉酶基因（AMY），并进行生物学信息分析及其表达规律研究，为揭示AMY基因的生物学功能及其对罗氏沼虾生长发育的调控作用机理提供理论依据。【方法】PCR克隆罗氏沼虾AMY基因编码区（CDS）序列，利用ExPASy ProtParam、ExPASy ProtScale、SignalP-5.0、MegAlign及Lasergenev8.0等在线软件进行生物信息学分析，应用实时荧光定量PCR检测AMY基因在罗氏沼虾不同组织（腹神经节、胃、心脏、鳃组织、性腺、肝胰腺、肌肉和肠道）中的表达情况，并明确其在生长快速家系（FG）和生长慢速家系（SG）个体肌肉中的表达模式。【结果】罗氏沼虾AMY基因CDS序列全长2121 bp，共编码706个氨基酸残基；其编码蛋白分子量为76.87 kD，理论等电点（pI）为4.63，属于不稳定的亲水性蛋白，包含2个典型的淀粉酶结构域；在罗氏沼虾AMY蛋白二级结构中，α-螺旋占13.88%，延伸链占21.39%，无规则卷曲占64.73%。罗氏沼虾AMY氨基酸序列与克氏原螯虾AMY氨基酸序列的相似性最高（62.6%），与大珠母贝AMY氨基酸序列的相似性较低（48.9%）；基于AMY氨基酸序列相似性构建的系统发育进化树显示，罗氏沼虾与克氏原螯虾的亲缘关系最近，而与中华绒螯蟹的亲缘关系较远。AMY基因在罗氏沼虾不同组织中广泛表达，但存在性别差异性和组织特异性，具体表现为:雌性个体肠道中的相对表达量极显著高于雄性个体（P<0.01，下同），鳃组织中的相对表达量显著高于雄性个体（P<0.05），而肝胰腺中的相对表达量极显著低于雄性个体。罗氏沼虾AMY基因在FG个体肌肉中的相对表达量极显著高于SG个体。【结论】AMY基因在罗氏沼虾的生长发育过程中发挥重要作用，广泛表达于罗氏沼虾的不同组织中，但存在性别差异性和组织特异性。
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| 关 键 词： | 罗氏沼虾 淀粉酶基因（AMY） 表达规律 生物信息学分析 生长性状 |
| 收稿时间： | 2020-05-23 |
Cloning of amylase gene in Macrobrachium rosenbergii and its expression regulation |
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| JIANG Jian-ping,YANG Xue-ming,YUAN Xiang,QIU Qing-qing,YANG Xiu-rong,JIANG Qin-yang,HUANG Guang-hua,JIANG He-sheng.Cloning of amylase gene in Macrobrachium rosenbergii and its expression regulation[J].Journal of Southern Agriculture,2021,52(5):1362-1369. |
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| Authors: | JIANG Jian-ping YANG Xue-ming YUAN Xiang QIU Qing-qing YANG Xiu-rong JIANG Qin-yang HUANG Guang-hua JIANG He-sheng |
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| Affiliation: | 1. Guangxi Botanical Garden of Medicinal Plants, Nanning 530023, China;2. College of Animal Science and Technology, Guangxi University, Nanning 530004, China;3. Guangxi Academy of Fishery Sciences, Nanning 530021, China |
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| Abstract: | 【Objective】Macrobrachium rosenbergii amylase gene(AMY) was cloned, biological information analysis and expression regulation were studied, to provide theoretical foundation for further understanding biological function of AMY gene in the growth and development of M. rosenbergii.【Method】The coding area sequence(CDS) of AMY gene in M. rosenbergii was cloned by PCR. Bioinformatic analysis of AMY sequencing was performed using ExPASy ProtParam, ExPASy ProtScale, SignalP-5.0, MegAlign and Lasergene v8.0 softwares. Real-time fluorescence quantitative PCR(RTPCR) was used to detect the expression of AMY gene in different tissues(including abdominal ganglion, stomach, heart, gill, gonad, hepatopancreas, muscle and intestine), and further to explore its expression in the muscle of individuals from fast-growing families(FG) and slow-growing families(SG).【Result】The length of CDS of AMY gene was 2121 bp, encoding 706 amino acids residues. The molecular weight of AMY protein was 76.87 kD, and the theoretical isoelectric point (pI) was 4.63, belonging to an unstable hydrophilic protein, which contained two typical amylase domains. The secondary structure of AMY protein was consisted of α-helix, extended strand and random coil, accounting for 13.88%, 21.39% and 64.73%, respectively. The homology of amino acid sequence of AMY gene in M. rosenbergii was the highest(62.6%) and lower(48.9%) compared with the amino acid sequences of Procambarus clarkii and Pinctada maxima, respectively. Phylogenetic tree constructed based on the amino acid sequence similarity of AMY genes howed that M. rosenbergii had the closest genetic relationship with P. clarki, but had the far genetic relationship with Eriocheir sinensis. AMY gene was widely expressed in the different tissues, but with differences between sexes and tissue specificity. The details were as follows:it had the extremely higher expression in the intestines and gills of female individuals than that of male individuals (P<0.01, the same below), it had the significantly higher expression in gill of female individuals than that of male individuals(P<0.05), but the expression in the hepatopancreas of female individuals was lower than that of male individuals. In addition, the expression level of AMY gene in muscle of in FG was extremely higher than that in SG.【Conclusion】AMY gene plays an important role in growth and development of M. rosenbergii. It is widely expressed in different tissues of M. rosenbergii, but with sex specificity and tissue specificity. |
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