异柠檬酸脱氢酶IDH的乙酰化调控及对黑胸散白蚁分飞行为的影响

Swarming behavior has important significance for dispersal and distribution of termites, and level of energy supply is an important factor influencing termite swarming behavior. Our previous research work found that there was an acetylation locus K143 in isocitrate dehydrogenase (IDH) from alates of Reticulitermes chinensis, and that acetylation level of K143 in IDH was significantly down-regulated after alate flight. However, it is still unknown that how IDH acetylation takes part in regulating the process of energy metabolism associated with swarming behavior of termites. This study will use deacetylase inhibitor NAM and TSA to treat with wild-type, and positive and negative mutant Sf9 cells containing acetylation locus K143 in IDH, and then will analyze the change of acetylation level and catalyzing activity of IDH so as to verify key acetylation locus regulating activity of IDH. We will clone and express two deacetylase genes SIRT3 and SIRT5 in alates of R. chinensis, and then will analyze the regulating effect of SIRT3 and SIRT5 on acetylation level and catalyzing activity of IDH in wild-type and mutant Sf9 cells. We will also investigate the influence of sensitive deacetylase and deacetylase inhibitor on acetylation, ATP level, respiratory control ratio and flight ability of alates in R. chinensis. These results are helpful to reveal the mechanism of energy supply of termite swarming behavior, and will provide new thought for developing energy inhibitor so as to control alate flight and damage of termites.

分飞行为对白蚁的扩散和分布具有重要意义，而能量供应水平又是影响白蚁分飞行为的一个重要因素。前期研究工作发现，黑胸散白蚁分飞蚁体内异柠檬酸脱氢酶IDH存在乙酰化位点K143，而且分飞后IDH乙酰化水平显著下调，但是IDH乙酰化修饰如何参与调控白蚁分飞行为的能量代谢过程尚不清楚。本项目利用去乙酰化酶抑制剂NAM和TSA处理IDH乙酰化位点K143的野生型和正负突变型Sf9细胞，分析IDH乙酰化水平和催化活性的变化，确定IDH活性调节的关键乙酰化位点。对黑胸散白蚁分飞蚁体内去乙酰化酶SIRT3和SIRT5基因进行克隆与表达，在不同突变Sf9细胞中分析SIRT3和SIRT5对IDH乙酰化水平和催化活性的调控作用。分析敏感去乙酰化酶及其抑制剂对分飞蚁IDH乙酰化修饰、ATP水平、呼吸控制比和飞行能力的影响。研究结果有助于揭示白蚁分飞行为的能量供应机制，为开发白蚁分飞能量抑制剂控制白蚁危害提供新思路。

分飞配对行为对白蚁的扩散和定殖至关重要。有翅蚁在一定的环境条件下可以使它们的分飞同步进行，这有利于随后的配对、远缘杂交和生殖。赖氨酸乙酰化和琥珀酰化在某些生理过程中起着重要作用，但其是否以及如何影响分飞及后续的交配选择尚不清楚。为此，本课题测定分析了黑胸散白蚁有翅蚁在分飞前后定量蛋白质乙酰化及琥珀酰化组学。结果表明，有翅蚁分飞后，在乙酰化组和琥珀酰化组中鉴定到6个乙酰化水平上升的蛋白，43个乙酰化水平下降的蛋白和55个琥珀酰化下调的蛋白，主要富集在能量代谢酶、飞行肌结构和内稳态调节三个方面。在sf9细胞中发现赖氨酸乙酰化可以改变TCA循环关键代谢酶IDH的催化活性，蛋白质乙酰化的变化减少了分飞蚁的能量供应，抑制了分飞蚁的随机飞行冲动，从而触发有翅蚁的同步分飞。副性腺蛋白（ACP）是有翅蚁分飞后唯一蛋白表达量、乙酰化水平和琥珀酰化水平都显著改变的蛋白，ACP的乙酰化和琥珀酰化修饰共同参与调控了有翅蚁分飞后串联追逐的对象选择。由此可见，蛋白质乙酰化和琥珀酰化修饰是操纵白蚁分飞同步性和配对选择性的一个重要表观遗传调控机制。