酪氨酸羟化酶TH基因和多巴脱羧酶DDC基因对拟黑多刺蚁品级分化调控机制的研究

Tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) belong to the key enzymes for synthesis of dopamine (DA) in mammals and insects, which play important roles in the regulation of mammalian and insect growth development and physiological activities. Usually, DA can influence insect ecdysone synthesis. The ecdysone as one of the insect critical hormones possesses abilities in regulating insect growth and metamorphosis especially in the ecdysis. When the ecdysone combing with ecdysone receptor (EcR), a cascade reaction of the insect molt will be set off and finally completes the insect molt. EcR, as the only nuclear receptor possessing ligand in insects till now, owns the typical nuclear receptor function domains and is similar to the estrogen receptor-related receptor (ERR). ERR belongs to the third nuclear receptor subfamily (NR3), and is identified as the first orphan nuclear receptor. ERR is also an important hormone in regulating human, vertebrate and invertebrate animals including insect growth and development especially in reproduction aspects. .Ecdysone can influence the social insect castes differentiation. Whether the TH and DDC genes directly effect the caste differentiation, or through impacting ecdysone synthesis to affect the insect caste differentiation, then regulating ecdysone receptor and other hormone receptors such as the estrogen receptor-related receptor, these are all unclear. So it is necessary to research the mechanism of TH and DDC gene regulating the insect caste differentiation and the association with the EcR or other hormone receptor. In this study, we based on the cloned TH and DDC genes from Polyrhachis vicina Roger, investigating the TH and DDC genes mRNA and protein expression levels in the ant body covering different castes and different development stages, probing into TH and DDC genes relevance with the ecdysone receptor (EcR) and estrogen receptor-related receptor (ERR) genes in the differentiation of reproductive individual and non reproductive individual, confirming the regulation mechanism of TH and DDC genes on social insect caste differentiation. The main methods include real time fluorescence quantitative PCR, hybridization in situ, immunohistochemistry, Western-blot and RNA interference technology. This research firstly carry out the regulation mechanism of TH and DDC genes on social insect caste differentiation in the domestic. The research of the mechanism of social insect castes differentiation may possess very important significance for further knowing the genetic mechanism of the insects division of social organization, understanding the animal and human social origin as well as evolution pathway.

酪氨酸羟化酶（TH）及多巴脱羧酶（DDC）是哺乳类和昆虫体内参与合成多巴胺(DA)的两种关键酶，对调控哺乳类和昆虫生长发育等生理活动起重要作用。TH、DDC基因是否对社会性昆虫品级分化有调控作用？还是通过影响蜕皮激素和其它激素及其受体对昆虫品级分化起调控作用，不清楚。因此有必要开展TH、DDC基因对社会性昆虫品级分化影响和调控机制的研究。本项目拟以已克隆的拟黑多刺蚁TH、DDC基因为基础，通过对其在该虫体内表达变化情况及与蜕皮激素受体（EcR）、雌激素相关受体（ERR）基因关联性研究，证实TH、DDC基因对品级分化的调控机制。本研究主要采用实时荧光定量PCR、原位杂交、免疫组织化学、Western-blot、RNA干扰等技术。本研究属于国内首次开展TH、DDC基因对社会性昆虫品级分化调控机制的研究。其成果对认识昆虫社会组织分工遗传学机制，了解动物和人类社会性成因及演化路径具有非常重要意义。

酪氨酸羟化酶（Tyrosine hydroxylase，TH）及多巴脱羧酶（Dopa decarboxylase，DDC）是哺乳类及昆虫体内参与合成多巴胺(Dopamine，DA)的两个关键酶，对调控哺乳类及昆虫的生长发育起着重要作用。我们前期已克隆这两个基因的cDNA，发现这两个基因在拟黑多刺蚁不同品级虫体均有mRNA差异性表达，由此推测可能与其对此种昆虫品级分化调节有关，但未得到证实。本项研究以之前对PvTH、PvDDC的研究为基础，对这两个基因在拟黑多刺蚁不同发育时期虫体及不同品级成虫mRNA及蛋白质表达进行RNA干扰前后的定量定位检测及分析比较；对PvTH、PvDDC与PvERR（雌激素相关受体，已克隆）及PvEcR（蜕皮激素受体，已克隆）关联性进行探究。确定PvTH、PvDDC对拟黑多刺蚁品级分化及生长发育的调控机制；确定PvTH、PvDDC与PvERR及PvEcR之间的关联性及这些基因对调控此种蚂蚁品级分化及生长发育所起的应有作用。.研究发现PvTH及PvDDC对拟黑多刺蚁品级分化及虫体发育具有重要的调控作用。对与成虫品级分化相关的学习记忆、嗅觉、感觉及运动控制等神经系统发育及性腺等生殖系统发育具有重要的调节作用；对幼虫及蛹的发育至关重要。PvTH基因对成虫与品级分化密切相关的脑神经系统结构及生殖系统结构的特点具有重要调控作用；PvDDC对成虫与品级分化密切相关的表型塑造、运动器官形成、生殖生理活动、脂类代谢、免疫功能及虫体寿命具有的重要调控作用。.研究发现PvTH、PvDDC与PvERR及PvEcR之间存在着关联性，这四个基因相互作用，对拟黑多刺蚁品级分化及虫体的发育具有重要调控作用。PvTH被干扰后，对PvERR及PvEcR的表达有影响，对两者的影响具有相对一致性，侧重点有所不同。PvDDC被干扰后，对PvEcR及PvERR的表达有影响，对两者的影响具有相似性。PvERR与PvTH基因之间的关联性明显比PvERR与PvDDC基因的关联性密切。PvERR与PvTH之间可能存在着双向调节。PvEcR对PvTH的影响力与PvEcR对PvDDC的影响力一样重要，差异不明显。PvEcR与PvTH之间可能存在着双向调节；PvEcR与PvDDC之间也可能存在着双向调节。.本项研究为探索动物乃至人类的性别分化及社会行为的发生及发展奠定了良好的研究基础。