Cloning and expression analysis of serine protease gene in Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) | 桔小实蝇丝氨酸蛋白酶基因（BdorSer）的克隆与表达分析

中国人. 克隆桔小实蝇丝氨酸蛋白酶基因（Bactrocera dorsalis serine protease, BdorSer），研究BdorSer在不同组织和不同发育时期的表达情况，探索其可能参与的生理过程。采用反转录聚合酶链式反应(RT-PCR)和快速扩增cDNA末端(RACE)技术克隆BdorSer，对其编码蛋白氨基酸序列信息和进化关系进行分析；采用实时荧光定量PCR（real-time PCR）方法，研究BdorSer mRNA在桔小实蝇不同组织及不同发育时期的相对表达量。克隆获得了BdorSer，该基因ORF全长1 239 bp。氨基酸序列一致性分析表明，BdorSer与拟暗果蝇（Drosophila pseudoobscura）丝氨酸蛋白酶（登录号XP_001352960）氨基酸序列的一致性最高，为65.8%。实时荧光定量PCR分析表明，BdorSer mRNA在桔小实蝇雌雄虫的不同组织中都有表达，且在触角中的表达量最高，分别是雌虫胸部的43.6和26.8倍。BdorSer mRNA在生殖节中表现出较大的性别差异表达特征，雄虫生殖节中的mRNA表达量是雌虫的18.25倍。BdorSer mRNA几乎在昆虫发育的各个时期都有表达，其中在卵及1，2，3龄幼虫中的表达量分别是10 d 蛹的 0.68，1.63，4.31和15.8倍。BdorSer mRNA随蛹的发育其表达量逐渐减低， 1，4，7 d蛹中的表达量分别是10 d蛹的325，125和41倍。克隆获得了BdorSer，其表达的BdorSer蛋白可能在雄虫的生殖生理中发挥了作用，同时也可能参与了桔小实蝇的变态发育过程，尤其是1 d蛹的发育过程。

英语. The study explored the possible participation of Bactrocera dorsalis (Hendel) serine protease gene (BdorSer) in physiology processes, cloned the cDNA and analyzed its expression pattern in all tissues and development stages. Reverse transcription PCR (RT-PCR) and Rapid amplification cDNA ends (RACE) method were used to clone BdorSer gene. The temporal and spatial expression levels of BdorSer mRNA were investigated by real-time quantitative RT-PCR method. The sequencing results showed that the full-length of open reading frame in BdorSer is 1 239 bp in length. The putative amino acid shared the highest similarity(65.8%) with XP_001352960, serine protease from Drosophila pseudoobscura. Real-time PCR results indicated that BdorSer expressed in all tissues while the highest expression level of BdorSer was detected in female and male antenna, the expression level of which was about 43.6 and 26.8 folds of that in female thorax, respectively. The amount of BdorSer mRNA in male genital segment was about 18.25 folds of that in female counterpart, apparently sex-biased expression pattern. BdorSer mRNA was also detected in all life cycle. The expression level in egg, 1, 2 and 3-d-old instar was about 0.68, 1.63, 4.31 and 15.8 folds of that in the 10-d-old pupae, which reveals a gradual upward trend. However, the expression level of BdorSer gradually decreased in the whole pupa stage. The expression level in 1, 4 and 7-d-old pupa was about 325, 125 and 41 folds of that in 10-d-old pupa. BdorSer gene was cloned. BdorSer plays an important role not only in the male reproductive physiological process but also in the metamorphosis process, especially in the 1-d-old pupa stage.