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利用实时荧光定量PCR法测算红螯螯虾实体组织的细胞数量
郑在超,李钫,杨丰
0
(自然资源部第三海洋研究所、海洋生物遗传资源重点实验室,福建 厦门 361005)
摘要:
螯虾是重要的水产养殖动物,也是研究甲壳动物疾病与免疫的一个良好模型。在研究不同组织的功能及受病原感染的影响时,需要了解组织内的细胞数量。但是,目前常用的细胞计数方法仅适用于单细胞悬液,无法用于实体组织。本研究以红螯螯虾(Cherax quadricarinatus)为对象,建立了基于基因拷贝数定量分析的实体组织细胞数量测算方法。首先克隆了beta-actin基因和甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase)基因(GAPDH)的部分DNA序列。在其内含子和外显子区域分别设计引物,使之只能以基因组DNA为模板进行扩增。其次,通过对实时荧光定量PCR (qPCR)熔解曲线的评估,确定以beta-actin作为目标基因。以血细胞基因组DNA为标准模板,进行beta-actin基因的qPCR分析。结果表明,在1×10~1×105个细胞的范围内,Ct值与细胞数量的对数之间呈良好的线性关系。线性回归得到回归曲线和回归方程Ct=38.30-3.37lg (N),N为细胞数量。最后,取红螯螯虾的鳃和肝胰腺组织为待测样,进行beta-actin基因qPCR分析,得到Ct值;根据上述方程计算得鳃组织细胞含量为1.8×105~2.2×105个细胞/mg组织,而肝胰腺组织细胞含量为5.5×105~6.0×105个细胞/mg组织。这一基于基因拷贝数和qPCR的细胞定量方法,具有简单便捷和高通量的优点,可用于测算螯虾实体组织的细胞数量。
关键词:  海洋生物学  细胞计数  红螯螯虾  实时荧光定量PCR  甲壳动物
DOI:10.3969/J.ISSN.2095-4972.2021.02.017
基金项目:国家自然科学基金资助项目(31672691, 31672675)
Determination of the cell number in different tissues of Cherax quadricarinatus by real-time fluorescence quantitative PCR
ZHENG Zai-chao,LI Fang,YANG Feng
(Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, MNR, Xiamen 361005, China)
Abstract:
Crayfish is an important aquaculture animal and a good model for the research of crustacean diseases and immunity. When studying the function of different tissues and the effects of pathogen infection, it is necessary to know the number of cells in the tissue. However, most of the cell counting methods are only applicable to single-cell suspensions, which are not suitable for solid tissues. In this study, we established a method to determine the cell number in the tissues of Cherax quadricarinatus basing on quantitative analysis of gene copy number. Firstly, partial DNA sequences of two genes, beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned. For qPCR analysis, one primer was designed in exon and the other was designed in intron. Secondly, the qPCR melt curves were analyzed for both genes, and beta-actin was chosen as the target gene. Crayfish hemocytes were counted by cell counter and used as the standard samples for qPCR analysis. The results showed that the Ct value of beta-actin gene has a linear correlation with the number (N) of hemocytes in range of 1×101-1×105cells. The linear regression equation is Ct=38.30-3.37lg (N). Finally, to determine the cell number in gill and hepatopancreas, DNA was extracted from these tissues. The beta-actin gene was analyzed by qPCR, and the cell numbers were calculated with the formula. The number of cells in gill was (1.8-2.2)×105 cells/mg tissue, and the number in hepatopancreas was (5.5-6.0)×105 cells/mg tissue. This simple and high-throughput quantitative method greatly facilitates the cell counting in solid tissues.
Key words:  marine biology  cell counting  Cherax quadricarinatus  qPCR  crustacean

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