两种色型黄粉虫酚氧化酶原的cDNA克隆、生物信息学分析及表达水平检测  

Cloning, bioinformatic analysis and expression profiling of the prophenoloxidase cDNA from two color varieties of Tenebrio molitor (Coleoptera:Tenebrionidae)

作　　者：黄琼[1] 胡杰[2] 王勤[3] 

机构地区：[1]四川农业大学林学院森林保护学省级重点实验室,四川雅安625014 [2]四川农业大学经济管理学院,成都611130 [3]四川农业大学生命科学与理学院生物系,四川雅安625014

出　　处：《昆虫学报》2013年第6期594-604,共11页Acta Entomologica Sinica

基　　金：四川省教育厅资助项目(10ZA056;07ZZ022);国家级大学生创新性实验计划资助项目(101062624)

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�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）. Tran

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 

分 类 号：Q966[生物学—昆虫学]