【摘要】 酚氧化酶是黑色素合成和昆虫免疫的关键酶,通常以无活性的酚氧化酶原形式存在。为给黄粉虫Tenebrio molitor遗传分化和免疫防御研究提供参考,本研究采用PCR和RACE技术克隆了黄、黑两种色型黄粉虫幼虫的酚氧化酶原基因Tm-ppo,对其cDNA序列及其推导的氨基酸序列进行生物信息学分析,采用实时荧光定量PCR检测其在两种色型黄粉虫不同发育阶段mRNA表达水平上的动态变化。结果表明:从黄、黑两种色型黄粉虫幼虫中克隆出的两个Tm-ppocDNA序列全长均为2199bp,碱基序列一致性为99%,包含一个2055bp的开放阅读框,编码684个氨基酸,它们的编码蛋白有3个氨基酸差异:第176位(P→A)、256位(V→A)和648位(I→M)。这两个基因分别被命名为Tm-ppo-1和Tm-ppo-2(GenBank登录号分别为JX987235和JX987234)。Tm-ppo-1和Tm-ppo-2编码的酚氧化酶原异构体蛋白(分别为Tm-PPO-1和Tm-PPO-2)存在一个可能的酚氧化酶原水解活化位点(R50～F51残基之间)和一个双铜结合中心(第196～239位残基和第344～411位残基);同时含有一个类似巯基酯区域的序列(第579～588位残基)及一个C-末端保守基序(第634～645位残基);但它们无氨基端疏水信号肽序列,也不存在跨膜区域。Tm-PPO-1和Tm-PPO-2的二级结构由大量的α-螺旋、β-折叠和无规则卷曲组成;三级结构分为前导域(第16～66位残基)、不相邻的功能域Ⅰ(第3～15位残基和第67～181位残基)、功能域Ⅱ(第182～419位残基)和功能域Ⅲ(第420～679位残基)4部分。此外,Tm-ppo-1和Tm-ppo-2在黄、黑两种色型黄粉虫的各发育期均有表达,并且不同发育阶段的mRNA表达水平呈现明显的变化规律:幼虫期﹥成虫期﹥蛹期。同时,环境温度对Tm-ppo-1和Tm-ppo-2的mRNA表达具有显著影响:与常温对照组(25～30℃)相比,42℃暴露24h和48h的两种色型黄粉虫幼虫、蛹和成虫的mRNA表达量明显下调。相同试验条件下,黑色型黄粉虫幼虫和成虫的Tm-ppo-2mRNA表达量明显高于黄色型幼虫和成虫的Tm-ppo-1mRNA表达量,但两种色型黄粉虫蛹的Tm-ppo异构体表达量无显著差异。本研究为进一步探讨黄粉虫的遗传分化和免疫防御提供了有益参考。更多还原

【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 2199 bp in length. Their base sequence identity is 99% . Both contain a 2055 bp open reading frame encoding 684 amino acid residues. Because of three amino acid variation ( P 176 →A 176 , V 256 →A 256 and I 648 →M 648 ) 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 R 50 and F 51 ,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.更多还原