RNA m6A甲基化在蜜蜂级型分化中的功能研究

The caste differentiation in honeybee is a typical epigenetic phenomenon, which has been attracting great attentions from researchers. Diverse insights into revealing its mechanism have been arising, but the study in RNA methylation is limited. . In this study, the N6-methyladenosine (m6A) differentiation is profiled in honeybee larvae between queen and worker at the age of 1, 3 and 5 day using MeRIP-seq technique to investigate the roles of m6A in larvae development. Further, the transcriptome with different methylation in the two caste is analyzed to explore the crucial genes in the caste differentiation. These candidate genes are subsequently studied in function by expression suppressed using RNAi with micro-injection of honeybee larvae. Their functions are identified through the alteration of the destiny of operated larvae approved by juvenile hormone level. As a result, the roles of crucial genes in the caste differentiation will be well understand and their mechanism will be revealed. Similar methods are carried out to interfere the genes of RNA m6A methylase, investigating the regulation roles of m6A methylation in the caste differentiation. . This research is an original design that the caste differentiation is systematically studied through RNA m6A methylation analysis. It will definitely provide a new insight to uncover this mystery in honeybee.

蜜蜂级型分化作为典型的表观遗传现象，是蜜蜂生物学研究的热点问题，但针对其RNA修饰的研究却鲜有报道。.本研究利用MeRIP-seq技术，结合免疫沉淀和高通量测序，比较意大利蜜蜂蜂王与工蜂1、3、5日龄幼虫6-甲基腺嘌呤（m6A）甲基化差异，确定RNA m6A甲基化在幼虫发育过程中的关键作用；分析比较幼虫发育关键时期蜂王和工蜂幼虫m6A甲基化差异转录本，筛选与蜜蜂级型分化相关的关键基因；通过蜜蜂卵显微注射的RNAi技术，干涉候选基因，并结合体内保幼激素滴度变化观测幼虫级型分化方向，确定目标基因在级型分化中的关键作用并揭示调控机制；采用相同的RNAi技术对蜜蜂m6A甲基化酶基因进行干涉，探讨m6A甲基化修饰在蜜蜂级型分化中的调控作用。.本研究首次系统开展RNA m6A甲基化在蜜蜂级型分化中的功能分析，为揭开蜜蜂级型分化科学谜题提供了新的契机。

在级型分化过程中，遗传物质相同的雌性蜜蜂幼虫，因食物不同而发育为可育蜂王和不育工蜂两个迥异的级型。DNA甲基化对级型分化的调控已被报道。RNA的N6-甲基腺苷（m6A）是真核生物中最丰富的RNA修饰，并能够可逆的调控基因表达，但是，m6A是否在级型分化中发挥功能性作用尚不清楚。本项目中，我们对不同日龄的蜂王和工蜂幼虫进行了m6A表观转录组（MeRIP-seq）分析，发现m6A甲基化被差异饲喂所改变。幼虫发育过程中，m6A甲基化修饰丰度随幼虫日龄增加而显著提高，并负向调节基因表达。与蜂王相比，工蜂幼虫具有更高的m6A甲基化水平。在蜂王和工蜂之间差异的m6A修饰转录本中，筛选出多个被m6A调控的级型分化相关基因，其中包括已知的级型分化调节因子，例如ILP-2，p110，PI3K和JHAMT等。另有被广泛研究但并未报道与级型分化相关的卵黄蛋白基因（Vitellogenin），其转录本显示出工蜂幼虫特有的高m6A修饰。本项目揭示了m6A甲基化在蜜蜂级型分化和幼虫发育中发挥了重要的调控作用。首次发现营养物质的摄入可以改变生物体内的m6A甲基化水平。这些发现将加深我们对社会性昆虫级型分化的认知，并拓宽蜜蜂研究的应用领域，使蜜蜂成为一种优异的表观遗传学模型，应用于动物发育及社会行为的研究中。