›› 2013, Vol. 56 ›› Issue (6): 594-604.

• 研究论文 • 上一篇    下一篇


黄琼1,*, 胡杰2, 王勤3   

  1. (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)
  • 出版日期:2013-06-20 发布日期:2013-06-20

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

HUANG Qiong1*, HU Jie2, WANG Qin3   

  1. (1. Provincial Key Laboratory of Forest Protection, College of Forestry, Sichuan Agricultural University, Ya’an, Sichuan 625014, China; 2. College of Economics and Management, Sichuan Agricultural University, Chengdu 611130, China; 3. Department of Biology, College of Life and Basic Sciences, Sichuan Agricultural University, Ya’an, Sichuan 625014, China)
  • Online:2013-06-20 Published:2013-06-20

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-ppo cDNA序列全长均为2 199 bp, 碱基序列一致性为99%, 包含一个2 055 bp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24 h和48 h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-2 mRNA表达量明显高于黄色型幼虫和成虫的Tm-ppo-1 mRNA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。

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