棘腹蛙Japonicin-pb抗菌肽的分离及表达谱分析

Isolation and Expression Patterns of Japonicin-pb from Paa boulengeri

本试验旨在克隆棘腹蛙来源Japonicin-pb,了解该抗菌肽在不同生长温度及组织下的表达规律,为Japonicin-pb的开发提供线索。基于本实验室构建的棘腹蛙皮肤转录组数据库,利用反转录PCR从棘腹蛙皮肤组织中克隆Japonicin-pb前体序列,通过结构域比对的方法分析其结构。利用Real-time PCR检测3种Japonicin-pb在不同生长温度(15、18、21、24、27和30℃)及组织(血液、肌肉、肝脏和皮肤)的表达图谱。结果表明,本试验克隆到3种Japonicin-pb前体序列,结构域比对表明,三者拥有相同的N端信号肽和中间间隔区,长度分别为19、20和12个氨基酸残基的肽序列,命名为Japonicin-1apb、Japonicin-1bpb和Japonicin-3pb。Japonicin-1apb主要在皮肤组织中表达,且表达量随着生长温度的升高而逐渐增加;Japonicin-1bpb在不同组织或生长温度下的表达量差异均不明显;Japonicin-3pb主要在肝脏中表达,且生长温度对其影响不大。这些结果为棘腹蛙源3种Japonicin-pb后续功能研究以及开发应用提供了重要的序列及表达谱信息。

In order to provided clues to the development of Japonicin-pb, we cloned the Japonicin-pb from Paa boulengeri and investigated its expression patterns among different growth temperatures and tissues. The com- plete open reading frame （ORF） sequence of Japonicin-pb precursor was amplified by RT-PCR according to the transcriptome database of Paa boulengeri constructed by our laboratory. The sequential structure of Japoni- cin-pb was analyzed by the method of domain alignment. The expression patterns of Japonicin-pb among differ- ent growth temperatures （15, 18, 21, 24, 27 and 30 ℃ ） and tissues （blood, muscle, liver and skin） were analyzed by real-time PCR. The results showed that according to the domain alignment, 3 Japonicin-pb precur- sors had the same N terminal signal peptide, middle spacer region, but the mature peptides were 19, 20 and 12 amino acid residues, named Japonicin-lapb, Japonicin-lbpb and Japonicin-3pb, respectively. The expression patterns showed that Japonicin-lapb was mainly expressed in skin, and its expression abundance increased gradually from 15 to 30 ℃ ; the expression level of Japonicin-lbpb among different tissues and growth temper- atures were no significant difference; Japonicin-pb mainly distributed in liver, and it was not sensitive to the response of growth temperature. These results provide important clues for the functional study and the develop- ment of Japonicin-pb.