意大利蜜蜂蜂王与工蜂幼虫甲基供体S-腺苷甲硫氨酸合成与代谢的差异

Differences of Methyl Donor S-adenosylmethionine Synthesis and Metabolism Between Apis mellifera ligustica Queen Bee Larvae and Worker Bee Larvae

【目的】研究意大利蜜蜂(Apis mellifera ligustica)蜂王与工蜂幼虫甲基供体S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)合成与代谢的差异,为探索DNA甲基化与蜜蜂级型分化的关系提供理论依据。【方法】试验选用890只1日龄雌性蜂幼虫,分别来自5群姐妹蜂王群。其中445只进行人工育王(89只/群);剩余445只培育成工蜂(89只/群)。取3、4、5日龄蜂王和工蜂幼虫,测定其体内SAM合成与代谢关键酶基因表达和酶活性的差异。【结果】蜂王幼虫SAM含量随日龄的增加变化不显著(P>0.05);工蜂幼虫SAM含量随日龄增加呈上升趋势(P<0.05)。蜂王幼虫SAMS基因表达量随日龄增加呈梯度下降的趋势(P<0.01),而工蜂幼虫SAMS表达随日龄变化不显著(P>0.05);3日龄与4日龄时,蜂王幼虫SAMS表达量显著高于工蜂(P<0.05),5日龄时,工蜂幼虫SAMS表达量显著高于蜂王(P<0.05)。Dnmt1a与Dnmt3表达在两级型间差异不显著(P>0.05),其中蜂王幼虫Dnmt1a表达随日龄增加无显著变化(P>0.05),但其酶活性呈下降趋势(P<0.05);工蜂幼虫Dnmt1a表达随日龄增加呈下降趋势(P>0.05),其酶活性呈上升趋势(P<0.01),其中3日龄与4日龄时,蜂王幼虫Dnmt1酶活性显著高于工蜂幼虫(P<0.05),而5日龄时,工蜂幼虫Dnmt1酶活性显著高于蜂王幼虫(P<0.05)。蜂王Dnmt3表达量随日龄增加呈下降趋势(P<0.05),工蜂幼虫Dnmt3表达随日龄增加变化不显著(P>0.05);蜂王幼虫Dnmt3酶活性随日龄变化不显著(P>0.05),工蜂幼虫Dnmt3酶活性随日龄变化显著(P<0.05),但蜂王幼虫Dnmt3酶活性在3、4、5日龄均显著高于工蜂幼虫(P<0.01)。【结论】3—5日龄意大利蜜蜂蜂王幼虫与工蜂幼虫体内活性甲基供体SAM的合成与代谢存在差异。在4日龄之前,蜂王幼虫SAM的合成比工蜂活跃,4日龄之后,工蜂幼虫的SAM合成与蜂王幼虫相近;在4日龄之前,SAM参与DNA维持甲基化的代谢过程,蜂王幼虫比工蜂活跃,4日龄之后,工蜂幼虫比蜂王幼虫活跃;在3—5日龄,SAM参与DNA从头甲基化的代谢活性,蜂王幼虫始终不低于工蜂幼虫。

【Objective】 The objective of this study is to research the differences of synthesis and metabolism of the methyl donor S-adenosylmethionine（SAM） between Apis mellifera queen bee and worker bee larvae, so as to provide a theoretical basis for the study of DNA methylation and caste differentiation. 【Method】 A total of 890 1-day-old female bee larvae selected from 5 colonies of sister queen group, respectively. Among the 890 larvae, 445 larvae were bred to the king（89 in each colony） and the other 445 larvae were put in the original group so that they can develop toward the worker caste naturally（89 in each colony）. The 3-, 4- and 5-day-old queen bee and worker bee larvae were taken to measure the differences of gene expression and enzyme activities of the key enzymes of SAM biosynthesis and metabolism in vivo.【Result】The content of SAM in queen bee larvae didn＇t change significantly with the increased days of age（P〈0.05）. The SAM content of worker bee larvae increased with the increase of days（P〈0.05）. The gene expression of SAMS in queen bee larvae showed a gradient descent trend with increasing age（P〈0.01）; but the gene expression of SAMS in worker bee larvae did not change significantly with the increase of days of age（P〈0.05）. The gene expression of SAMS in queen bee larva was higher than worker bee larvae at 3- and 4-day-old obviously（P〈0.05）, but the gene expression of SAMS in worker bee larva was higher than queen bee larvae obviously at 5-day-old（P〈0.05）. The gene expression of Dnmt1 a and Dnmt3 was not different significantly between the two types（P〈0.05）. The gene expression of Dnmt1 a in queen bee larvae showed no significant change with the increase of days of age（P〈0.05）, but the enzyme activity of Dnmt1 in queen bee larvae decreased with the increase of days of age（P〈0.05）. The gene expression of Dnmt1 a was decreased with the increase of days of age in worker bee larvae（P〈0.05）, but the enzyme activity of Dnmt1 showed a rising trend（P〈0.01）. The enzyme activity of Dnmt1 in queen bee larvae was higher than the worker bee larvae significantly at 3-, 4-day-old（P〈0.05）, but the enzyme activity of Dnmt1 in worker bee larvae was higher than the queen bee larvae significantly at 5-day-old（P〈0.05）. The gene expression of Dnmt3 in queen bee larvae showed a decreasing trend with the increased days of age（P〈0.05）, the gene expression of Dnmt3 in worker bee larvae showed no significant change with the increased days of age（P〈0.05）. The enzyme activity of Dnmt3 changed in queen bee larva was not significant with the increased days of age（P〈0.05）, the enzyme activity of Dnmt3 changed significantly in worker bee larvae with the increased days of age（P〈0.05）, but the enzyme activity of Dnmt3 in queen bee larvae were higher significantly than that of worker bee larvae at 3-, 4- and 5-day-old（P〈0.01）. 【Conclusion】There were some differences of the methyl donor（SAM） synthesis and metabolism between queen bee larvae and worker bee larvae at 3-, 4- and 5-day-old. Synthesis of SAM in queen bee larvae was more active than the worker bee larvae before 4-day-old and close to worker bee larvae after 4-day-old. The metabolism process of SAM participating in DNA maintenance methylation in queen bee larvae were more active than worker bee larvae before 4-day-old, but lower than worker bee larvae after 4-day-old. The metabolism process of SAM participating in de novo methylation of DNA in queen bee larva was not lower than worker bee larvae from 3- to 5-day-old.