缢蛏糖原磷酸化酶基因多态性与糖原含量的关联分析

陈燕园; 林志华; 刘圣; 姚韩韩; 董迎辉

doi:10.12284/hyxb2022068

缢蛏糖原磷酸化酶基因多态性与糖原含量的关联分析

doi: 10.12284/hyxb2022068

陈燕园^{1, 2,},
林志华^{1, 3},
刘圣³,
姚韩韩^1, ,,
董迎辉¹

1.
浙江万里学院生物与环境学院浙江省水产种质资源高效利用技术研究重点实验室，浙江宁波 315100
2.
上海海洋大学水产科学国家级实验教学示范中心，上海 201306
3.
浙江万里学院宁海海洋生物种业研究院，浙江宁海 315604

基金项目: 国家重点研发计划“蓝色粮仓”科技创新课题（2018YFD0901405）；浙江省农业新品种选育重大科技专项（2021C02069-7）；国家现代贝类产业技术体系项目（CARS-49）；国家海洋水产种质资源库项目；宁波市“科技创新2025”重大专项（2019B10005）。

详细信息

作者简介:
陈燕园（1994—），福建省漳州市人，研究方向为贝类分子遗传与育种。E-mail: 1304397861@qq.com

通讯作者:
姚韩韩，高级实验师，研究方向为贝类分子遗传与育种。E-mail: yaohanhan1020@126.com

中图分类号: Q786；S917.4
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出版历程
- 收稿日期: 2021-06-07
- 修回日期: 2021-11-21
- 网络出版日期: 2021-12-25
- 刊出日期: 2022-02-01

The association of single nucleotide polymorphisms of the glycogen phosphorylase gene with glycogen content in the razor clam Sinonovacula constricta

Chen Yanyuan^{1, 2
,},
Lin Zhihua^{1, 3},
Liu Sheng³,
Yao Hanhan^{1
, ,},
Dong Yinghui¹

1.
Zhejiang Key Laboratory of Aquatic Germplasm Resources, College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China
2.
National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
3.
Ninghai Marine Biological Seed Industry Research Institute, Zhejiang Wanli University, Ninghai 315604, China

摘要

摘要: 为探究缢蛏糖原磷酸化酶基因（Sc-GPH）与糖原含量的关系，本研究克隆获得Sc-GPH cDNA全长，检测其在不同组织、不同月份的表达模式，并在Sc-GPH基因编码区筛选与糖原含量关联的SNP位点。结果表明，Sc-GPH cDNA全长为3 963 bp，开放阅读框为2 541 bp，编码846个氨基酸；氨基酸序列多重比对和系统发育树显示，缢蛏与欧洲大扇贝、虾夷扇贝、长牡蛎等贝类亲缘关系较近，而与哺乳类、甲壳类、昆虫类亲缘关系较远。实时荧光定量PCR结果显示，Sc-GPH在8个组织中均有表达，其中在外套膜和足中表达量最高（p<0.01），推测与其糖原存储能力有关；不同月份的缢蛏外套膜和足中，Sc-GPH在8月表达量最高，而此时糖原含量较低，说明Sc-GPH的表达可能受缢蛏生殖周期的影响。以缢蛏“甬乐1号”群体为实验材料，在Sc-GPH基因编码区筛选到4个与糖原含量相关SNP位点，其中c.930T>C位点在台州野生群体中得到进一步验证，该位点为高糖原缢蛏分子标记辅助选育提供了候选标记。
- 缢蛏 /
- 糖原磷酸化酶 /
- 糖原含量 /
- SNP /
- 关联分析
Abstract: To clarify the relationship between the glycogen phosphorylase gene (Sc-GPH) and glycogen content in Sinonovacula constricta, the full-length cDNA sequence of Sc-GPH is obtained using RACE technology, its expression pattern in different tissues, different months are analyzed, and the SNP loci of Sc-GPH associated with glycogen content are explored. The full-length cDNA of Sc-GPH gene is 3 963 bp and the ORF is 2 541 bp, encoding 846 amino acids. The multiple alignment of amino acid sequences and phylogenetic tree show that S. constricta are closely related to shellfish such as Pecten maximus, Mizuhopecten yessoens, Crassostrea gigas, but farther to mammals, arthropodas and insects. The results of real-time quantitative PCR show that Sc-GPH gene is expressed in eight tissues, with the highest expression in mantle and foot, suggesting they are related to glycogen storage capacity. In different months, the expression levels of Sc-GPH in the mantle and foot is the highest in August, at which time the glycogen content decreased, indicating that the expression of Sc-GPH seems to be driven by reproductive cycle of S. constricta. In “YL1” population, four SNPs related to glycogen content are screened in the coding region of Sc-GPH gene, and c.930T>C is further verified in Taizhou wild population, which provides a candidate marker for molecular marker assisted breeding of high glycogen S. constricta varieties.
- Sinonovacula constricta /
- glycogen phosphorylase /
- glycogen content /
- SNP /
- correlation analysis

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图 1 缢蛏不同组织（A）和不同月份足和外套膜（B）的糖原含量变化

1. 鳃；2. 闭壳肌；3. 足；4. 外套膜；5. 肝胰腺；6. 性腺；7. 水管；8. 唇瓣；A中不同小写字母代表差异极显著（p<0.01）；B中同一组织不同字母代表差异极显著（p<0.01）

Fig. 1 Changes of glycogen content in different tissues (A) and different months in foot and mantle (B) of Sinonovacula constricta

1. Gill; 2. adductor muscle; 3. foot; 4. mantle; 5. hepatopancreas; 6. gonad; 7. siphon; 8. palp; different lowercases indicate extremely significant difference (p<0.01) in A; different letters marked on columns of the same tissue represent their extremely significant differences in data (p<0.01) in B

下载: 全尺寸图片幻灯片

图 2 缢蛏与其他物种GPH氨基酸序列系统进化分析

进化树中各物种GPH氨基酸序列的GenBank登录号为：缢蛏（Sinonovacula constricta，MT125681）；长牡蛎（Crassostrea gigas，CCN27372）；欧洲大扇贝（Pecten maximus，XP_033734012）；虾夷扇贝（Mizuhopecten yessoensis，OWF50424）；克氏原螯虾（Procambarus clarki，AVN99053）；凡纳滨对虾（Penaeus vannamei，QCY50320）；日本对虾（Procambarus clarkii，BAJ23879）；异色瓢虫（Harmonia axyridis，ASZ80181）；黑腹果蝇（Drosophila melanogaster，NP_001027219）；家蚕（Bombyx mori，ACB41088）；甜菜夜蛾（Spodoptera exigua，ACN78408）；亚洲玉米螟（Ostrinia furnacalis，AFO54708）；智人（Homo sapiens，AAC18079）；小家鼠（Mus musculus，AAG00588）；马（Equus caballus，BAH22533）；单峰驼（Camelus dromedarius，KAB1277008）

Fig. 2 Neighbor-joining phylogenetic tree of GPH among Sinonovacula constricta and other species

GenBank accession numbers of GPH sequences used for phylogenetic tree: Razor clam （Sinonovacula constricta, MT125681）; Pacific oyster （Crassostrea gigas, CCN27372）; European scallop （Pecten maximus, XP_033734012）;Japanese scallop （Mizuhopecten yessoensis, OWF50424）; Red swamp crayfish （Procambarus clarki, AVN99053）; Whiteleg shrimp （Penaeus vannamei, QCY50320）; Kuruma prawn （Penaeus japonicus, BAJ23879）; Asian lady beetles （Harmonia axyridis, ASZ80181）; Fruit fly （Drosophila melanogaster, NP_001027219）; Domestic silkworm （Bombyx mori, ACB41088）; Beet armyworm （Spodoptera exigua, ACN78408）; Asiatic corn borer （Ostrinia furnacalis, AFO54708）; Human （Homo sapiens, AAC18079）; House mouse （Mus musculus, AAG00588）; Horse （Equus caballus, BAH22533）; Single hump camel （Camelus dromedarius, KAB1277008）

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图 3 Sc-GPH在缢蛏不同组织（A）和不同月份足和外套膜（B）的的表达特征

Fig. 3 The expression characteristics of Sc-GPH in different tissues (A) and different months in foot and mantle (B)

1. Gill; 2. adductor muscle; 3. foot; 4. mantle; 5. hepatopancreas; 6. gonad; 7. siphon; 8. palp; different lowercases indicate extremely significant difference（p<0.01）in A; different letters marked on columns of the same tissue represent their extremely significant differences in data （p<0.01）in B

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表 1 所用的引物及信息

Tab. 1 The information of primers used in this experiment

引物名称	引物序列（5′-3′）	用途
H3	AGTGAACAGATCTCCACCGCAGGCA	Sc-GPH 3′-RACE扩增
H5	GCGGGATGACTTGAACCTACGGAT	Sc-GPH 5′-RACE扩增
18S-F	TCGGTTCTATTGCGTTGGTTTT	qPCR
18S-R	CAGTTGGCATCGTTTATGGTCA	qPCR
H-Q-F	ATCTACAACTCCCTCCTCTACCA	qPCR
H-Q-R	GGCACATCTTAGCCCAAAG	qPCR
H-S-F	AAAGACATTCAATCGCCACCT	SNP筛选
H-S-R	TTTCTGGATTTGGCTCACAACCC	SNP筛选

下载: 导出CSV

表 2 缢蛏“甬乐1号”（YL1）和台州野生群体（TZ）Sc-GPH SNPs位点与糖原含量的相关性分析

Tab. 2 Association of SNPs of Sc-GPH with glycogen content in YL1 and TZ of Sinonovacula constricta

群体	SNP位点	基因型	样本量/频率	糖原含量/(mg·g⁻¹)	p值
YL1	c.874C>T	CC	89/0.89	54.6±30.3	0.047^*
		CT	11/0.11	74.1±30.0
	c.930T>C	TT	49/0.49	52.1±29.4	0.029^*
		TC	43/0.43	57.9±28.4
		CC	8/0.08	78.6±34.7
	c.1133 T>C	TT	87/0.87	53.6±29.2	0.023^*
		TC	13/0.13	77.7±27.1
	c.1284 T>A	TT	65/0.65	61.0±26.7	0.028^*
		TA	23/0.23	56.8±37.7
		AA	12/0.12	33.1±1.97
TZ	c.930T>C	TT	47/0.47	73.9±22.5	0.047^*
		TC	40/0.4	80.7±23.2
		CC	13/0.13	86.1±15.0
注：p值代表SNP位点与糖原含量的相关程度，*代差异显著（p<0.05）。

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