State Key Laboratory of Silkworm Genome Biology and College of Biotechnology, Southwest University, Chongqing, China ; College of Biotechnology, Southwest University, Chongqing, China.
PLoS One. 2013 Aug 26;8(8):e71984. doi: 10.1371/journal.pone.0071984. eCollection 2013.
Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4(th) instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.
昆虫发育过程中的色素沉着是一种原始的适应性需求。在蚕中，黑色素是幼虫色素的主要成分。黑色素合成的限速底物是酪氨酸，它由限速酶苯丙氨酸羟化酶（PAH）从苯丙氨酸转化而来。虽然酪氨酸（来自苯丙氨酸）在纤维蛋白合成中的作用早已为人所知，但 PAH 在黑色素合成中的作用在蚕中仍不清楚。为了确定 PAH 的重要性，我们克隆了 BmPAH 的 cDNA 序列，并使用 Bac-to-Bac 杆状病毒表达系统表达其完整的编码序列。纯化的重组蛋白具有高 PAH 活性、一些色氨酸羟化酶活性，但没有酪氨酸羟化酶活性，这是无脊椎动物中 PAH 的典型特性。由于黑色素合成在胚胎阶段和幼虫体壁重新着色阶段最为旺盛，我们将 BmPAH dsRNA 注入蚕卵中，观察到 BmPAH mRNA 减少会降低新生幼虫的酪氨酸含量，并导致昆虫变色失败。体外培养和 4 龄幼虫体壁注射 PAH 抑制剂表明，PAH 活性对于幼虫标记颜色至关重要。这些数据表明，BmPAH 是黑色素合成所必需的，我们提出 PAH 将苯丙氨酸转化为酪氨酸是蚕黑色素生物合成途径的第一步。
