Gene Cloning And Expression Analysis Of PTTH In Antheraea Pernyi

Antheraea pernyi is a unique insect in China. In order to fight against the harsh environment and keep population continue, A. pernyi stay diapause during the period of pupa. During the diapause period, the development of insects is temporarily stopped. After termination of diapause, the insects will continue to grow and reproduce. Prothoracicotropic hormone (PTTH) secreted by the insect brain, and stimulates the secretion of gland ecdysone to control insect metamorphosis. Researching PTTH of Antheraea pernyi can lead further understanding of the insect diapause mechanisms, and help artificially regulate diapause-termination in nature light environment in order to improve the high-production.The 5th larva, pupa and adult of Yuda No.1 (univoltine tussah variety from Henan province) and Shenhuang No.2 (bivoltine tussah variety from Shenyang province) were used in this experiment. Using RT-PCR technology, PTTH gene was successfully cloned from Antheraea pernyi. The structure and function of sequence was predicted using bioinformatics methods. ApPTTH prokaryotic expression vector was constructed and successfully expressed. Total RNA was isolated from the 5th stages of Shenhuang No.2 larva, and tissues of larvae as mentioned above were used for semi-quantitative PCR to detect expression. The pupa of univoltine and bivoltine tussah were stay under 300 Lux illuminance condition for 17 h. Using the real-time fluorescence quantitative PCR technology, ApPTTH gene expression were detected in the brain of Yuda No.1 and Shenhuang No.2 in diapause-determinated progress. The results were as follows:1) Using TA cloning technology, we successfully cloned ApPTTH gene:The ORF of ApPTTH gene sequence is 666 bp, encoding 221 amino acids. Blastp analysis revealed that the predicted amino acid sequence of ApPTTH shared more than 70% identity to the amino acid sequence of other insects.2) Semi-quantitative PCR technology detection:ApPTTH gene was only expressed in brain and ovarian during the different tissue of the 5th instar larval.3) The results of Real-time PCR detection:ApPTTH keep stabilization during the progress of diapause pupa, but changed a lot during the determination of diapauses and pre-adult. When coming to the adult, the expression level declined. This indicated that ApPTTH play an important role in diapause-breading and adult development.4) Construct a prokaryotic expression vector, the preparation of antibody and the Western-blot identification:Connect the the sequence with pEASY-Blunt E1 expression vector, and ApPTTH was expressed and purified for antibody preparation. Western-blot were used to detect PTTH in the brain of A. pernyi. This research laid a foundation for understanding ApPTTH function and diapause breaking.