TRANSCRIPTOME-BASED ANALYSIS OF OVIPAROUS AND OVOVIVIPAROUS-RELATED GENE EXPRESSION IN ARTEMIA
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摘要: 为揭示卤虫(Artemia)不同繁殖模式的发生机制, 文章通过构建孤雌生殖卤虫卵生和卵胎生差异转录组文库并结合生物信息学分析, 对两种繁殖模式间的差异表达基因进行查找筛选, 然后利用qRT-PCR对候选繁殖模式相关基因的表达进行分析。转录组测序显示有1452个差异表达基因, 包括601个上调基因和851个下调基因。根据差异表达基因GO功能分类结果可知, 注释到生物过程、细胞组成和分子功能的unigene分别有1243、306和530个。KEGG富集分析结果显示差异基因显著富集在抗原加工和核糖体通路中。结合转录组分析, 进一步筛选得到6个生殖相关基因, 并针对不同繁殖模式下的卤虫进行qRT-PCR, 结果表明, 6个生殖相关基因在卵生卤虫卵巢中的表达量均显著高于卵胎生卤虫。此外, 对6个候选生殖相关基因编码的蛋白质保守结构域进行预测, 发现均与之前报道的相应基因保守结构域一致。综上所述, 研究所选择的6个基因可能影响参与了卤虫的生殖过程。研究结果为孤雌卤虫繁殖模式分子机制调控的研究提供了基础信息, 有助于完善卤虫的生殖生物学理论。Abstract: Artemia is not only one of the most important live feed for larvi culture, but also an ideal experimental organism for scientific research. Female Artemia produce either nauplii via ovoviviparous pathway or diapause cyst via oviparous pathway. In order to reveal the mechanism of different reproductive modes of Artemia, the reproductive differential transcriptomes of parthenogenetic Artemia were constructed, bioinformatics analysis were performed to screen reproductive differential expression genes, and the gene expression patterns were studied by using qRT-PCR. Transcriptome analysis showed that there were 1452 differentially expressed genes, of which 601 genes were up-regulated and 851 down-regulated in the abscising carpopodium. According to GO function classification, 1243306 and 530 unigene were annotated into biological process, cell composition and molecular function respectively. KEGG enrichment analysis showed that differential genes were significantly enriched in antigen processing and ribosome pathways. Combined with transcriptome data and qRT-PCR analysis, six reproductive-related genes were screened and verified. The results showed that all the six reproductive-related genes had higher expression in oviparous Artemia than in ovoviviparous Artemia. In addition, the conserved domains of the proteins encoded by six candidate reproductive related genes were predicted and phylogenetic trees were constructed respectively. The results showed that the protein domains were consistent with the previously reported reproductive genes. Over all, our study indicated that the selected six genes may influence the reproductive process of Artemia. This study provides valuable information for dissecting the molecular mechanism of reproductive pattern in the parthenogenetic Artemia, and may also help to refine the reproductive biological theory of Artemia.
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Keywords:
- Transcriptome /
- Reproductive-related genes /
- Reproductive mode /
- Artemia
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图 1 卵胎生与卵生卤虫卵巢中上调和下调的差异表达基因
Figure 1. Up-regulated and down-regulated differentially expressed genes in ovoviviparous vs oviparous Artemia ovary
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图 4 SLK、Dmrt3、Fem-1、CyclinB、5-HT和Nanos蛋白保守结构域预测
A. SLK蛋白; B. Dmrt3蛋白; C. Fem-1蛋白; D. CyclinB蛋白; E. 5-HT蛋白; F. Nanos蛋白
Figure 4. Prediction of conservative domains of SLK, Dmrt3, Fem-1, CyclinB, 5-HT and Nanos proteins
A. SLK protein; B. Dmrt3 protein; C. Fem-1 protein; D. CyclinB protein; E. 5-HT protein; F. Nanos protein
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图 5 SLK、Dmrt3、Fem-1、CyclinB、5-HT和Nanos蛋白系统进化树
Figure 5. Phylogenetic tree of SLK, DMRT3, Fem-1, CyclinB, 5-HT and Nanos proteins
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图 6 SLK、Dmrt3、fem-1、cyclinB、5-HT和Nanos基因在不同繁殖模式下的表达情况
相同字母表示差异不显著(P>0.05); 不同字母表示差异极显著(P<0.01); OVI. 卵生; OVOVI. 卵胎生
Figure 6. Expression of SLK, Dmrt3, fem-1, cyclinB, 5-HT and Nanos genes in different reproductive modes of Artemia oocysts
The same letter indicate no significant difference (P>0.05); the difference between different letters is the extremely remarkable difference (P<0.01); OVI: oviparous; OVOVI. ovoviviparous
表 1 qRT-PCR引物
Table 1 qRT-PCR primers
引物Primer 序列Sequence (5′—3′) 5-HT-F GCACCTCGAATCCCTTGAAC 5-HT-R GAAATCTTGAGCGCCTCTGG cyclin B-F ACGCCACAGTCATGAAGAGA cyclin B-R TCTTCTCAAATGCGCAGACG Nanos-F TGGTGAAGCCGAATTGATGC Nanos-R TTTCTTTCAGCGCGTGACTT Dmrt3-F CTGCAACAGGAGCCTCAATC Dmrt3-R TGGAGTTGTAGTCGGAGTCG fem-1-F ACGCCTTAGAAATGCAGCAG fem-1-R TCACAGTCAAGACCCGTTGA SLK-F AAAGGCAACTCAAGCACCAG SLK-R ACTTCCCGTGAATGTCACCT β-actin-F GTGTGACGATGATGTTGCGG β-actin-R GCTGTCCTTTTGACCCATTCC 
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表 2 Unigene序列在公共数据库中的注释结果
Table 2 Comment results of the unigene sequence in the public database
数据库Database 注释基因数量
Number of
annotated genes注释百分比
Annotation
percentage (%)NR数据库 33184 34.15 NT数据库 12641 13.01 PFAM数据库 36049 37.10 KO数据库 20595 21.20 KOG/COG数据库 19946 20.53 Swiss-prot数据库 31480 32.40 GO数据库 36331 37.39 在所有数据库中均注释 4109 4.22 合计ALL 97146 100.00
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表 3 卤虫表达上调和下调基因富集的10个信号通路
Table 3 The top -ten enriched pathways of up -regulated and down-regulated unigene in Artemia
通路名称
Pathway_term富集因子
Rich_factorQ值
Q value基因数
Gene_
number上调表达基因
Up-regulated unigene蛋白质消化吸收
Protein digestion and absorption0.047619048 0.000168817 10 阿米巴疾病
Amoebiasis0.044025157 0.005670205 7 叶酸生物合成
Folate biosynthesis0.076923077 0.108059701 3 甘油脂代谢
Glycerolipid metabolism0.035714286 0.141920793 4 细胞外基质受体相互作用
ECM-receptor interaction0.027777778 0.141920793 5 谷胱甘肽代谢
Glutathione metabolism0.032786885 0.141920793 4 小细胞肺癌
Small cell lung cancer0.032520325 0.141920793 4 PI3K-Akt信号通路PI3K-Akt signaling pathway 0.017130621 0.141920793 8 化学致癌作用
Chemical carcinogenesis0.044776119 0.141920793 3 肾素-血管紧张素系统Renin-angiotensin system 0.044117647 0.141920793 3 下调表达基因
Down-regulated unigene核糖体
Ribosome0.063157895 4.68E-12 54 抗原处理和呈递
Antigen processing and presentation0.159722222 4.68E-12 23 长寿调节途径-多种生物
Longevity regulating pathway-multiple species0.081730769 5.20E-05 17 溶酶体
Lysosome0.063333333 0.00030973 19 军团菌病
Legionellosis0.077348066 0.000509087 14 胰岛素抵抗
Insulin resistance0.067961165 0.001609763 14 细胞凋亡
Apoptosis0.048192771 0.003953602 20 麻疹Measles 0.064327485 0.011564643 11 吞噬体
Phagosome0.044776119 0.014675213 18 弓形虫病
Toxoplasmosis0.057803468 0.034988835 10
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