关键词: 黄粉虫, 色型, 酚氧化酶原, 基因异构体, 生物信息学, mRNA表达

Abstract: Phenoloxidase is the key enzyme of melanin synthesis and insect immunity, usually existing in the form of prophenoloxidase. In order to study the genetic differentiation and immune defense, a prophenoloxidase gene Tm-ppo was cloned from the larvae of yellow and black color varieties of Tenebrio molitor, bioinformatics of the cDNA and the encoded amino acid sequence of Tm-ppo was analyzed, and the mRNA levels in different developmental stages of the two varieties were examined by PCR, RACE and realtime quantitative PCR. The results showed that the full cDNA sequences of Tm-ppo cloned from the yellow- and black-color varieties of T. molitor are both 2 199 bp in length. Their base sequence identity is 99%. Both contain a 2 055 bp open reading frame encoding 684 amino acid residues. Because of three amino acid variation (P176→A176, V256→A256 and I648→M648) existing between the two proteins encoded by the two cDNA sequences, they were considered two isoforms of Tm-ppo, i.e., Tm-ppo-1  (GenBank accession number: JX987235) and Tm-ppo-2 (GenBank accession number: JX987234), respectively. Both prophenoloxidase protein isoforms (Tm-PPO-1 and Tm-PPO-2) encoded by Tm-ppo-1 and Tm-ppo-2 have a possible prophenoloxidase proteolytic activation site located between the amino acid residues of R50 and F51, and a di-copper binding centre appearing at residues of 196-239 and residues of 344-411, respectively. In addition, they contain a thiol ester region-like motif (residues of 579-588) and a C-terminal conserved motif (residues of 634-645). But they have neither a hydrophobic N-terminal signal sequence nor a transmembrane domain. The secondary structure of Tm-PPO-1 and Tm-PPO-2 consists of many alpha helices, beta sheets and random coils, and their tertiary structure can be divided into 4 functional domains: the pro-region (residues of 16-66), the noncontiguous domain Ⅰ (residues of 3-15 and 67-181), domain Ⅱ (residues of 182-419) and domain Ⅲ (residues of 420-679). Transcripts of Tm-ppo-1 and Tm-ppo-2 were evidently present in every developmental stage of the yellow and blackcolor varieties of T. molitor and the mRNA levels in each stage varied in the descending order of larval stage﹥adult stage﹥pupal stage. Environmental temperature had a significant effect on the mRNA expression of Tm-ppo-1 and Tm-ppo-2. The mRNA levels were all downregulated in larvae, pupae and adults of both color varieties with exposure to 42℃ for 24 h and 48 h, compared with those in the control insects under 25-30℃. The Tm-ppo-2 mRNA level in larvae and adults of blackcolor variety was higher than the Tm-ppo-1 mRNA level in larvae and adults of yellow-color variety, while the mRNA expressed in pupae of both color varieties showed no significant difference under the same test conditions. This study may provide a useful reference to further inquire into genetic differentiation and immune defense in T. molitor.

Key words: Tenebrio molitor, color variety, prophenoloxidase, gene isoform, bioinformatics, mRNA expression