昆虫中多种乙酰胆碱酯酶基因的存在，使不同乙酰胆碱酯酶的表达谱和功能研究成为热点之一。家蚕也存在两种乙酰胆碱酯酶基因：Bm-ace1, Bm-ace2。由于家蚕特殊的经济价值，加之其基因组序列已经公布，家蚕已经作为一种模式生物被广泛运用于分子生物学及生物信息学研究中。本课题的研究重点主要利用荧光定量PCR、生化试验、生物信息学方法对家蚕两种乙酰胆碱酯酶基因的表达谱及生化特性进行系统地研究。主要内容及研究结果如下： 1）运用荧光定量PCR技术测定家蚕两种乙酰胆碱酯酶基因在不同发育阶段（1-5龄、蛹、成虫、卵）及同一龄期不同日龄的表达量。结果表明，Bm-ace2表达量在所有发育阶段都明显高于Bm-ace1，Bm-ace2/ Bm-ace1的比例为2.92-64.75，成虫阶段尤其雄虫的差异最大。这与已报道的农业昆虫结果相反，已有的研究表明在多种农业害虫中乙酰胆碱酯酶Ⅰ表达量明显高于乙酰胆碱酯酶Ⅱ。分析认为在对昆虫多种乙酰胆碱酯酶基因的进化选择上存在多样性。由于特殊的生存环境，家蚕选择了ace2，而其他害虫选择了ace1。两种基因在不同发育阶段各自的表达趋势有差异，Bm-ace1在1龄幼虫表达最高，卵期表达最低，其他阶段差异不明显；Bm-ace2在卵期表达最低，在雄虫表达高于幼虫和雌虫。在3龄不同日龄幼虫， Bm-ace1表达量高峰出现在龄期初期（1天）、龄期中间（3天）和龄期末（5天）；Bm-ace2的表达趋势为第1-3天逐天上升，第3-5天逐天下降。根据定量结果， Bm-ace2在3龄中的表达量一直高于Bm-ace1。Bm-ace2/Bm-ace1的比例存在动态变化，最高峰出现在第2天，之后逐渐下降，即两种酯酶基本表达量差量逐天减小。在家蚕头部、消化道、马氏管、丝腺、卵巢、精巢中，两种ace基因均有表达，并且两种基因在不同的组织中表达量存在差异。在头部中两种酶都高表达，Bm-ace1在丝腺中也较高，Bm-ace2在生殖器官（卵巢和精巢）中高表达。推测除主要的水解乙酰胆碱功能外，两种乙酰胆碱酯酶可能存在其他非水解酶的功能。另外，与不同龄期中的表达一致，Bm-ace2表达量明显高于Bm-ace1。在精巢中Bm-ace2/Bm-ace1比值最高，在头部比值最低。 2）运用家蚕1、3龄不同日龄试虫粗酶液测定AChE酶活力及Km、Vm生化参数测定。结果表明，三龄幼虫不同日龄试虫酶活力成动态变化，1-3天逐渐升高，3-5龄逐渐下降，这与Bm-ace2基因的mRNA表达量趋势一致，而与Bm-ace1基因不一致，进一步证明Bm-ace2基因在家蚕体内占主导作用。在运用双倒数曲线计算Km、Vm时，发现在1、3龄不同日龄试虫的双倒数曲线不一致。这表明不同日龄试虫的AChE混合酶液的蛋白组分明显不同，这一方面是由于粗酶液是两种酶的混合物，另一方面是由两种酶不同时间的动态变化引起的。试验结果表明，在存在多种乙酰胆碱酯酶基因的昆虫中，运用粗酶液进行酶动力学测定的传统方法不能够正确反应酶生化特性，必须将多种AChE酶分别纯化后测定其生化特性参数。 3）通过生物信息学软件TESS预测Bm-ace1 、Bm-ace2转录因子结合核心区域上转录因子结合位点。在Bm-ace1的启动子区，预测到37个转录因子结合位点，而Bm-ace2的启动子区预测到33个结合位点。在这些位点中，两个基因共同的结合位点只有15个。两个基因拥有各自独特的转录因子结合位点，推测这些不同结合位点上结合的转录因子可能是引起家蚕两个乙酰胆碱酯酶基因表达量差异的重要原因之一。

The existence of two acetylcholinesterase gene in insect has opened research areas on ace expression and function analysis. The domesticated silkworm, Bombyx Mori, also has two acetylcholinesterase genes: Bm-ace1, Bm-ace2. Here we mainly focused on the two ace genes’ expression profiling and biochemical characteristics by using real-time quantitative PCR, biochemical test and bioinformatics. The main contents and research results are as follows: 1) The expression levels of two ace genes in different developmental stages (larva 1-5, pupa, adult, egg) and different day of the same stage were estimated by real-time quantitative PCR. The results show that, the abundance of Bm-ace2 in the each development stage was significantly higher than that of Bm-ace1. The ratio of Bm-ace2 / Bm-ace1 was 2.92-64.75. This is inconsistent with the agricultural pests. In agricultural pests, expression of ace1 was higher than that of ace2. The expression of Bm-ace1 is the highest in the 1-instar larvae, whereas the lowest expression is at egg stage. Bm-ace2 expressed at lowest level at the egg stage, and at highest in the male adult. During the 3rd instar of silkworm, Bm-ace1 had a high expression in the day 1, 3, 5; Bm-ace2 expressed 1-3 day-by-day rise and 2-5 day-by-day fall. The expression of Bm-ace2 is also higher than Bm-ace1 in each day at 3rd instar. The ratio of Bm-ace2/Bm-ace1 had a dynamic trend during 3rd instar, the highest peak is at the day 2 and decreased at 2-5 days. In the insect head, enteron, Malpighian tubules, silk gland, ovary, testis, the two ace-specific expressions of both genes are different, indicating its function differences. The two ace genes are both highly expressed in the head. Bm-ace1 highly expressed in the silk gland, and Bm-ace2 is abundent in the ovary and testis. It shows that besides the major function of terminate synaptic transmission, the two AChE may exist their non- hydrolysis functions. Furthermore, the Bm-ace2 expression was significantly higher than Bm-ace1. The Bm-ace2/Bm-ace1 ratio is the highest in the testis and lowest in the head. 2) We used the 1st and 3rd instar silkworm to determinate the AChE enzyme activity and Km, Vm using the crude extract. The results showed that the enzyme activity changes day-by-day. For the 3rd instar, the enzyme activity increase day-by-day at day 1-3 and decrease day-by-day at day 2-5. This is consistent with the mRNA expression trend of Bm-ace2. We found that the double-reciprocal curves of AChE are different in the 1st and 3rd instars. It indicated that mixed enzyme solution of AChE is instabe. We reasoned that this might be caused by the dynamics of quantitative change. This results challenges the conventional method of estimating enzymatic activity using crude extract as an enzyme solution, as it is a mixture of AChE1 and AChE2. An efficient and simple method for separating different AChEs is necessary for reliable toxicological analyses. 3) The bioinformatics software TESS was used to predict Bm-ace1, Bm-ace2 transcription factor binding sites on the core transcription factor binding region. It shows that there 37 transcription factor binding sites for the 34 kinds of transcription factors for Bm-ace1. For Bm-ace2 33, there are 48 transcription factors binding sites. Of these sites, only 15 binding sites were shared by both two genes. Different binding sites were presented at the promoter regions of two different silkworm AChE genes. We argued that transcription factors binding at different binding sites were the intrinsic regulatory factors caused the differences in the expression of two silkworm ace genes.