Molecular characterization and expression of MAP2K1 gene in Hyriopsis cumingii
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摘要: 为研究MAP2K1 (MEK1) 基因在三角帆蚌 (Hyriopsis cumingii) 性别决定中的作用，采用cDNA末端快速克隆技术 (Rapid-amplification of cDNA ends, RACE) 克隆了MAP2K1基因序列，利用实时荧光定量分析比较MAP2K1基因在三角帆蚌6个组织 (性腺、闭壳肌、肝胰腺、鳃、外套膜、斧足)、早期发育阶段 (1—8月龄) 性腺和1—3龄雌雄性腺中的表达水平，利用原位杂交确定MAP2K1基因在2龄三角帆蚌性腺中的定位。结果显示，MAP2K1基因开放阅读框 (ORF) 长度为1 194 bp，编码397个氨基酸。MAP2K1基因在卵巢中高表达；早期发育阶段在2月龄表达量最高；1—3龄的表达结果显示，MAP2K1基因在卵巢中的表达量均高于同期精巢中的表达量 (P<0.05)。原位杂交结果显示，MAP2K1基因在雌性三角帆蚌的卵母细胞及卵子上有明显的杂交信号。RNAi结果显示，干扰MAP2K1基因的上游基因C-MOS后，下游基因MAP2K1在雌性中的表达下降了82.31%，在雄性中的表达下降了73.60%。推测MAP2K1基因可能参与三角帆蚌的卵巢发育过程，在三角帆蚌中属于偏雌性基因，其表达受C-MOS基因的影响。Abstract: In order to study the role of the MAP2K1 (MEK1) gene in the sex determination of Hyriopsis cumingii, we applied RACE (Rapid-amplification of cDNA ends) method to clone the MAP2K1 gene sequence. We conducted a real-time fluorescence quantitative analysis to compare MAP2K1 gene in six tissues (Gonads, adductor muscle, hepatopancreas, gills, mantle, foot), gonads at early developmental stage (1−8 month old) and the 1−3 years' level of expression in male and female glands of H. cumingii. We determined the location of MAP2K1 gene in the gonads of 2-year-old H.cumingii by in situ hybridization. The results show that the ORF region of MAP2K1 gene was 1 194 bp in length and encoded 397 amino acids. MAP2K1 gene was highly expressed in the ovary; the expression level was the highest at 2 months of age at early developmental stage; the expression results from 1−3 years of age show that the expression of MAP2K1 gene in the ovary was higher than that in the spermatozoa for the same period (P<0.05). The in situ hybridization results show that the MAP2K1 gene had a significant hybridization signal in the oocytes and eggs of female H. cumingii. RNAi results show that the expression of the downstream gene MAP2K1 gene decreased by 82.31% in females and 73.60% in males after interfering with the upstream gene C-MOS gene of MAP2K1 gene. In conclusion, MAP2K1 gene may be involved in the ovarian development process and is a female-biased gene in H. cumingii, and C-MOS gene affects its expression.
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Key words:
- Hyriopsis cumingii /
- MAP2K1 gene /
- Molecular characterization /
- Gene expression
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图 1 三角帆蚌MAP2K1基因核苷酸序列及氨基酸序列
Figure 1. MAP2K1 gene nucleotide sequence and amino acid sequence of H. cumingii
图 2 三角帆蚌MAP2K1基因二级、三级结构预测
Figure 2. Secondary and tertiary structure prediction of MAP2K1 gene in H. cumingii
图 3 三角帆蚌与其他物种MAP2K1基因的蛋白序列比对
Figure 3. Protein sequence comparison of MAP2K1 gene between H. cumingii and others
图 4 不同物种MAP2K1基因的氨基酸序列构建的NJ系统进化树
Figure 4. NJ phylogenetic trees constructed from amino acid sequences of MAP2K1 gene in different species
图 6 三角帆蚌MAP2K1基因在早期性腺组织中的表达
注：柱上不同字母表示存在显著性差异 (P<0.05) ；相同字母表示无显著性差异。
Figure 6. Expression of MAP2K1 in various tissues of female and male adults of H. cumingii
Note: Different letters indicate significant difference (P<0.05), while the same letters indicate insignificant difference.
图 7 MAP2K1基因在1—3龄三角帆蚌性腺中的相对表达
Figure 7. Relative expression of MAP2K1 gene in gonads ofH. cumingii of 1−3 years old
图 8 三角帆蚌MAP2K1基因在2龄三角帆蚌性腺原位杂交
注：a. 雌性阴性对照组；b. 雌性实验组；c. 雄性阴性对照组；d. 雄性实验组。
Figure 8. MAP2K1 in situ hybridization in gonads of 2-year-old H. cumingii
Note: a. Female of negative control group; b. Female of experimental group; c. Male of negative control group; d. Male of experimental group.
图 9 三角帆蚌C-MOS基因干扰后表达情况
Figure 9. Expression of C-MOS gene of H. cumingii after interference
图 11 三角帆蚌C-MOS基因干扰后MAP2K1基因表达情况
Figure 11. Expression of MAP2K1 gene of H. cumingii after C-MOS gene interference
表 1 本实验所需引物
Table 1. Primers for this study
引物名
Primer's name引物序列 (5'—3')
Primer sequence (5'−3')用途
PurposeK1-O TATTCCTACAGCAGACTCGTGATTG 3'RACE outer K1-I GACCTGGATTTGATAATAGATGTGGG 3'RACE inner K1-YG-F AACAAGCTGAATCTGACGCTG 荧光定量正向引物 K1-YG-R GCTCCTTCAGTTTCTTGGTCAGT 荧光定量反向引物 EFl-αF GGAACTTCCCAGGCAGACTGTGC 内参正向引物 EFl-αR TCAAAACGGGCCGCAGAGAAT 内参反向引物 K1-YW-F AACAAGCTGAATCTGACGCTG 原位杂交正向引物 K1-YW-R TAATACGACTCACTATAGGGGCTCCTTCAGTTTCTTGGTCAGT 原位杂交反向引物 M-GR-1F TTGGGTATGGTTCGCAGTT 干扰链1正向引物 M-GR-1R GCCCTTGTAAGCTCATGTTT 干扰链1正向引物 T7+M-GR-1F TAATACGACTCACTATAGGGTTGGGTATGGTTCGCAGTT T7+干扰链1正向引物 T7+M-GR-1R TAATACGACTCACTATAGGGGCCCTTGTAAGCTCATGTTT T7+干扰链1反向引物 M-GR-2F TATGAACGAACATCGGAGGC 干扰链2正向引物 M-GR-2R CCCATACGCAACAACCC 干扰链2反向引物 T7+M-GR-2F TAATACGACTCACTATAGGGTATGAACGAACATCGGAGGC T7+干扰链2正向引物 T7+M-GR-2R TAATACGACTCACTATAGGGCCCATACGCAACAACCC T7+干扰链2反向引物 M-GR-3F TAACGCCTCACGACGAC 干扰链3正向引物 M-GR-3R CCGGCTCTTGCATTCC 干扰链3反向引物 T7+M-GR-3F TAATACGACTCACTATAGGGTAACGCCTCACGACGAC T7+干扰链3正向引物 T7+M-GR-3R TAATACGACTCACTATAGGGCCGGCTCTTGCATTCC T7+干扰链3反向引物 
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