EXPRESSION AND FUNCTIONAL ANALYSIS OF THE VITELLINOGEN GENE IN DAPHNIA SINENSIS
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摘要:
为了阐明卵黄蛋白原基因对溞属枝角类生殖和胚胎发育的影响机制, 研究了RNA干扰下中华拟同形溞(Daphnia sinensis)的卵黄蛋白原基因表达及产幼溞数变化。结果表明, RNA干扰后, 与EGFP (Enhanced green fluorescent protein)组相比, 2%大肠杆菌浓度下中华拟同形溞在Vtg组的首次产幼溞数和Vtg基因相对表达量没有显著差异; 5%大肠杆菌浓度下中华拟同形溞前四个成龄在Vtg组的产幼溞数均显著小于EGFP组, 且中华拟同形溞的第一成龄、第四成龄时在Vtg组的Vtg基因相对表达量均显著小于EGFP组, 分别下降了5.25%和78.03%。此外, RNA干扰后Vtg组的中华拟同形溞还出现了胚胎消解和后代发育不全的现象。结果表明, 高强度、长时间的RNA干扰能够显著降低中华拟同形溞Vtg基因的相对表达水平, 也能够显著抑制其生殖量和胚胎发育。因此, Vtg基因对中华拟同形溞的生殖和胚胎发育起重要作用。
Abstract:In order to elucidate the mechanism of the influence of vitellinogen gene on the reproduction and embryonic development of Daphnia, the changes on the expression of vitellogenin gene and the number of offspring of D. sinensis under RNA interference (RNAi) was studied. After RNA interference, compared with the EGFP (Enhanced Green Fluorescent Protein) group, there was no significant difference in number of offspring at the first reproduction or relative expression level of Vtg gene of D. sinensis in the Vtg group at the 2% E. coli concentration. At the 5% E. coli concentration, the number of offspring at the first four adult instar in the Vtg group were significantly lower than those in the EGFP group. Additionally, the relative expressions of the Vtg gene of D. sinensis at the first and the fourth adult instars in the Vtg group were significantly lower than those in the EGFP group, with decreases of 5.25% and 78.03%, respectively. In addition, the phenomenon of embryonic digestion and underdevelopment of offspring also appeared in the Vtg group after RNAi. The results suggest that high-intensity and prolonged RNA interference can significantly reduce the relative expression of the Vtg gene in D. sinensis, leading to a marked inhibition of reproductive capacity and embryonic development. Therefore, the Vtg gene plays an important role in the reproduction and embryonic development of D. sinensis.
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Keywords:
- Vtg gene /
- Reproduction /
- Functional analysis /
- Daphnia sinensis
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图 1 部分甲壳动物Vtg基因的氨基酸序列的系统进化树
Figure 1. Phylogenetic tree on amino acid sequence of Vtg gene in partial crustaceans
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图 2 dsRNA诱导图
M. DNA分子量标准Marker; 1—2. 分别携带L4440-Vtg、L4440-EGFP重组质粒的HT115菌株的诱导产物; 3. 携带L4440重组质粒的HT115菌株的未诱导产物
Figure 2. The induction of double strand RNA
M. DNA molecular weight standard; 1—2. Induced product of HT115 strain carrying recombinant plasmid of L4440-Vtg and L4440-EGFP, respectively; 3. HT115 strain carrying L4440 recombinant plasmid is not induced
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图 3 RNA干扰后中华拟同形溞的首次怀卵数、首次产幼溞数及第一成龄Vtg基因的相对表达量
Figure 3. Number of eggs at first pregnancy and offspring at the first reproduction and relative expression of mRNA of Vtg gene at the 1st adult instar of D. sinensis after RNAi
(2% and 5% stand for 2% and 5% E. coli concentrations, respectively; *P<0.05, **P<0.01)
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图 4 RNA干扰后中华拟同形溞的胚胎解体及后代畸形的现象
1. 显示正常发育的胚胎Showing normal embryo; 2. 显示消解的胚胎Showing disintegrated embryos; 3. 显示正常发育的后代Showing normal offspring; 4. 显示畸形的后代Showing abnormal offspring
Figure 4. The phenomenon of embryonic disintegration and offspring malformation of D. sinensis after RNAi
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图 5 5%大肠杆菌浓度下RNA干扰后中华拟同形溞前四个成龄的产幼溞数
Figure 5. Offspring number of D. sinensis at the first four adult instar after RNAi under 5% E. coli concentration (*P<0.05, ***P<0.001)
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图 6 5%大肠杆菌浓度下第四成龄时中华拟同形溞Vtg基因的表达水平
Figure 6. Expression levels of Vtg gene of D. sinensis at the 4th adult instar under 5% E. coli concentration (***P<0.001)
表 1 引物的序列及名称
Table 1 Sequence and name of primer employed in this experiment
引物名称
Primer name引物
Primer (5′—3′)GAPDH TCGTCTCCAATGCTTCTT
CGGTCCATCAACAGTCTTVtg CTTGGAAGGCAATGGAATCCCA
GGATGTGGATCAGGACATTATTGC
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