| 黄金凤，李 波，白 莹，冯礼燊，李文笙，孙彩云.吉富罗非鱼胃肠道几丁质酶的
克隆、组织分布和纯化[J].广东农业科学,2021,48(6):107-115 |
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吉富罗非鱼胃肠道几丁质酶的
克隆、组织分布和纯化 |
| cDNA Cloning, Distribution and Purification of Chitinases from Gastrointestinal Tract of GIFT Tilapia（Oreochromis niloticus） |
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| DOI：10.16768/j.issn.1004-874X.2021.06.015 |
| 中文关键词: 几丁质酶 吉富罗非鱼 组织分布 胃肠道 几丁质酶活 |
| 英文关键词: chitinase GIFT tilapia tissue distribution gastrointestinal tract chitinase activity |
| 基金项目:国家现代农业产业技术体系专项 ( CARS-46) ；广东省科技计划项目（2014A020208021）；广东省自
然科学基金面上项目（2019A1515011884） |
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| 中文摘要: |
| 【目的】几丁质酶是重要的水解酶，能通过水解 β-1,4- 糖苷键来降解鱼类食物中虾蟹壳所含的
几丁质，帮助鱼类消化。了解几丁质酶在罗非鱼不同组织中的表达状况，以及从罗非鱼胃中所提取几丁质酶的
特性。【方法】从吉富罗非鱼胃和肠组织中克隆获得 3 种几丁质酶 tChit1a、tChi3 和 tChit 的开放阅读框序列进
行序列分析，并通过 Real-time PCR 检测其组织分布状况；通过几丁质亲和层析法纯化罗非鱼胃组织中的几丁质
酶，并通过 4-MU 法检测酶活性。【结果】tChit1a、tChi3 和 tChit 几丁质酶基因分别编码 453、453 和 473 个氨
基酸，同源比对结果显示 tChit1a 与 tChi3 的相似度为 83.66%，而 tChit 与 tChit1a、tChi3 的相似度则较低，分别
为 49.89% 和 50.11%。进化树分析结果显示，这 3 种几丁质酶是鱼类三型几丁质酶，为非酸性几丁质酶。组织
分布结果显示，tChit1a、tChi3 和 tChit 分别在罗非鱼中肠、前肠和中肠后表达量最高。纯化罗非鱼胃几丁质酶
的结果显示，SDS-PAGE 胶在 40 ku 附近有两条明显条带，但用斜带石斑鱼 1 型几丁质酶多克隆抗体检测，没有
检测到同源性高的条带。检测纯化产物几丁质酶的活性，结果显示，在最适 pH 值为 5 时，纯化产物降解 4MU-
（GlcNAc）2 和 4MU-（GlcNAc）3 分别为 1.73、4.89 U/g。【结论】罗非鱼胃组织中所提取的几丁质酶表达量和
活性均较低，这可能与罗非鱼为杂食且偏素食的食性有关。 |
| 英文摘要: |
| 【Objective】Chitinases are the important hydrolytic enzymes, which can degrade the shells of shrimp and
crab containing chitin in fish food by hydrolyzing β-1,4- glycosidic bonds. In this study, we tried to under the expression
profile of chitinases in different tissues of tilapia and the characteristics of chitinase extracted from the stomach of tilapia. 【Method】Three chitinases, named tChit1a, tChi3 and tChit, were cloned from the stomach and intestines of GIFT tilapia.
ORFs of these chitinases were analyzed by a series of bioinformatics software and their tissue distribution were detected by
Real-time PCR. Next, the chitinases from stomach of tilapia were purified by affinity chromatography and the chitinase activity
was detected by the 4-MU method.【Result】The genes tChit1a, tChi3 and tChit encoded 453, 453 and 473 amino acids,
respectively. Homology alignment results showed that the homology between the deduced amino acid sequences of tChit1a
and tChi3 was 83.66%, while tChit showed lower homology to tChit1a and tChi3, with 49.89% and 50.11%, respectively. The
phylogenetic analysis indicated that tChit1a, tChi3 and tChit were classified into fish chitinases-3（FCase-3）rather than acid
fish chitinase-1/2（AFCase-1/2）. The result of tissue distribution showed that tChit1a, tChi3 and tChit mainly expressed in
the gastrointestinal tract of tilapia, with the highest mRNA expression in the midgut, foregut and midgut hind respectively. The
purified chitinases from stomach of tilapia showed that there were two obvious bands with the size of about 40 ku, but no high
homologous bands（Chitinase-1 of tilapia）were detected by polyclonal antibody of the Chitinase-1 of Epinephelus obliquus.
The activities of hydrolyzed 4MU-（GlcNAc）2
and 4MU-（GlcNAc）3
were 1.73 and 4.89 U/g respectively at the optimum
pH 5.【Conclusion】The low expression and activity of chitinases extracted from stomach of tilapia suggest that tilapia may
have little interest in ingesting chitinous substances |
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