Cloning and expression analysis of PCNA in Metapenaeus affinis
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摘要: 增殖细胞核抗原 (Proliferating cell nuclear antigen, PCNA) 作为DNA聚合酶δ的辅助蛋白，在DNA复制过程中发挥重要作用。近缘新对虾 (Metapenaeus affinis) 卵巢发育过程中存在细胞增殖旺盛活动的阶段，但目前关于其卵巢发育的分子机制研究依旧较少。利用RACE (Rapid amplification of cDNA ends) 技术获得近缘新对虾PCNA (MaPCNA) 的cDNA序列全长，并通过实时荧光定量PCR (qRT-PCR) 对其进行卵巢发育相关的表达分析。MaPCNA全长为1 144 bp，包含140 bp的5'非编码区，221 bp的3'非编码区，开放阅读框 (ORF) 为783 bp，编码260个氨基酸。MaPCNA蛋白的相对分子质量为28.82 kD，理论等电点为4.5。多重比对分析表明，PCNA氨基酸序列在甲壳动物中较为保守。组织表达结果显示，MaPCNA在检测的组织中均有表达，其中在卵巢表达量最为显著 (P<0.05)。在不同发育阶段的卵巢中，MaPCNA的表达出现变化 (P<0.05)，从I期开始逐渐上升，到III期表达量最高，之后显著降低并趋于平稳。MaPCNA在幼体发育不同时期的表达中呈现出一定的规律性趋势，在受精卵时期的表达量最高，之后呈下降趋势，从无节幼体VI期开始表达平稳。研究结果提示PCNA基因可能在近缘新对虾的卵巢发育过程中发挥重要作用。Abstract: As a coprotein of DNA polymerase δ, proliferating cell nuclear antigen (PCNA) plays an important role in the process of DNA replication. There is a stage of vigorous cell proliferation during the ovarian development in Metapenaeus affinis, but little attention has been paid to its molecular mechanism. In this study, we applied rapid amplification of cDNA ends (RACE) technique to obtain the full length cDNA sequences of PCNA in M. affinis (MaPCNA), and analyzed the expression of MaPCNA related to ovarian development by real-time fluorescent quantitative PCR (qRT-PCR). The total length of MaPCNA was 1 144 bp, including 140 bp of 5' untranslated region, 221 bp of 3' untranslated region and 783 bp ORF encoding 260 amino acids. The molecular weight of MaPCNA protein was 28.82 kDa and the theoretical isoelectric point was 4.5. The protein homology analysis shows that MaPCNA had high homology with other crustaceans. The result of tissue expression shows that MaPCNA was expressed in all tested tissues, with the highest expression in ovary (P<0.05). The expression of MaPCNA in ovaries at different developmental stages showed significant changes (P<0.05), increasing from stage I to stage III gradually, decreasing significantly and then tended to be stable. The expression of MaPCNA shows a regular change trend in different larval developmental stages. The expression level of MAPCNA was the highest in the oosperm, then began to decline, and became stable from the Nauplius VI. The results suggest that PCNA may play an important role in the ovarian development of M. affinis.
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Key words:
- Metapenaeus affinis /
- PCNA /
- Cloning /
- Gene expression
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图 2 MaPCNA与其他物种的PCNA基因之间的多重序列比对图
Figure 2. Multiple sequence alignment between MaPCNA and PCNA gene of other species
图 4 基于氨基酸序列比较的PCNA在不同物种中的系统进化树
Figure 4. Phylogenetic tree of PCNA in different species based on amino acid sequence comparisons
图 5 MaPCNA基因在不同组织中的相对表达量
注：图中数值为“平均值±标准差”(N=3)，小写字母不同表示各组之间具有显著差异 (P<0.05)；后图同此。
Figure 5. Relative expression of MaPCNA in different tissues
Note: The values are “X±SD” (N=3). Different lowercase letters indicate significant difference among groups (P<0.05); the same case in the following figures.
图 6 MaPCNA基因在在卵巢发育不同阶段的相对表达量
Figure 6. Relative expression of MaPCNA in different ovarian developmental stages
图 7 MaPCNA在幼体发育各阶段的相对表达量
Figure 7. Relative expression of MaPCNA in different larval developmental stages
表 1 实验所使用的引物序列
Table 1. Oligonucleotide primers used in experiment
引物
Primer引物序列 (5'—3')
Primer sequence (5'−3')用途
FunctionMaPCNA-F GGAAGTCTGTTGAAGAAGGTGTTGGA 验证序列 MaPCNA-R GTATTCTGCCACAAAGCCATAGTAAGC MaPCNA-5GSP1 TTTGGACATGCTGGTGAGGTTCATGCCC 5'RACE MaPCNA-5GSP2 CATGCCCATGATGAGGTTTCGGTCGC MaPCNA-3GSP1 AAAGCAACACCCCTTTCCCCACAGG 3'RACE MaPCNA-3GSP2 TCCCTGTCCATGTCTCCTGATGTACCCC UPM-long CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT RACE UPM-short CTAATACGACTCACTATAGGGC RACE NUP AAGCAGTGGTATCAACGCAGAGT MaPCNA-qF TCATCGAGATGCAGGAGCCAGTTA qPCR MaPCNA-qR ATCAGGAGACATGGACAGGGAGAC EF-1α-qF AAGCCAGGTATGGTTGTCAACTTT EF-1α-qR CGTGGTGCATCTCCACAGACT 
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