Long Yaohang, Li Jiaorong, Zhao Tianfu, Li Guannan, Zhu Yong
State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University, Number 2 Tiansheng Road, Beibei, Chongqing, 400715, China.
Appl Biochem Biotechnol. 2015 Apr;175(7):3447-57. doi: 10.1007/s12010-015-1516-3. Epub 2015 Feb 25.
Dopamine is a precursor for melanin synthesis. Arylalkylamine N-acetyltransferase (AANAT) is involved in the melatonin formation in insects because it could catalyze the transformation from dopamine to dopamine-N-acetyldopamine. In this study, we identified a new AANAT gene in the silkworm (Bombyx mori) and assessed its role in the silkworm. The cDNA of this gene encodes 233 amino acids that shares 57 % amino acid identity with the Bm-iAANAT protein. We thus refer to this gene as Bm-iAANAT2. To investigate the role of Bm-iAANAT2, we constructed a transgenic interference system using a 3xp3 promoter to suppress the expression of Bm-iAANAT2 in the silkworm. We observed that melanin deposition occurs in the head and integument in transgenic lines. To verify the melanism pattern, dopamine content and the enzyme activity of AANAT were determined by high-performance liquid chromatography (HPLC). We found that an increase in dopamine levels affects melanism patterns on the heads of transgenic B. mori. A reduction in the enzyme activity of AANAT leads to changes in dopamine levels. We analyzed the expression of the Bm-iAANAT2 genes by qPCR and found that the expression of Bm-iAANAT2 gene is significantly lower in transgenic lines. Our results lead us to conclude that Bm-iAANAT2 is a new arylalkylamine N-acetyltransferase gene in the silkworm and is involved in the metabolism of the dopamine to avoid the generation of melanin.
多巴胺是黑色素合成的前体。芳基烷基胺N - 乙酰基转移酶（AANAT）参与昆虫体内褪黑素的形成，因为它可以催化多巴胺向多巴胺 - N - 乙酰多巴胺的转化。在本研究中，我们在蚕（家蚕）中鉴定出一个新的AANAT基因，并评估了其在蚕中的作用。该基因的cDNA编码233个氨基酸，与Bm - iAANAT蛋白具有57%的氨基酸同一性。因此，我们将该基因命名为Bm - iAANAT2。为了研究Bm - iAANAT2的作用，我们构建了一个使用3xp3启动子的转基因干扰系统，以抑制家蚕中Bm - iAANAT2的表达。我们观察到转基因品系的头部和体表出现黑色素沉积。为了验证黑化模式，通过高效液相色谱（HPLC）测定了多巴胺含量和AANAT的酶活性。我们发现多巴胺水平的升高会影响转基因家蚕头部的黑化模式。AANAT酶活性的降低会导致多巴胺水平的变化。我们通过qPCR分析了Bm - iAANAT2基因的表达，发现转基因品系中Bm - iAANAT2基因的表达显著降低。我们的结果使我们得出结论，Bm - iAANAT2是家蚕中一个新的芳基烷基胺N - 乙酰基转移酶基因，参与多巴胺的代谢以避免黑色素的产生。
