家蚕追寄蝇孵化酶的基因克隆与特性分析

Gene Cloning and Characteristic Analysis of Hatching Enzyme from Exorista sorbillans

家蚕追寄蝇寄生于家蚕幼虫引起的蝇蛆病危害蚕业生产。为从分子水平研究家蚕追寄蝇的孵化机制,以发育1.5 d的蝇卵为材料,结合RACE技术获得家蚕追寄蝇孵化酶基因的全长cDNA,命名为EsHE(GenBank登录号:ON042475)。EsHE基因cDNA全长1 507 bp,包含159 bp 5′UTR、412 bp 3′UTR和936 bp的完整ORF,编码311个氨基酸;EsHE蛋白序列含有孵化酶特征序列HELMHVLGFMHE(锌结合基序)和YDYGSVMHY(Met-转角基序);系统进化树分析显示,家蚕追寄蝇与丝光绿蝇的孵化酶亲缘关系最近。qRT-PCR检测EsHE基因在家蚕追寄蝇卵期的表达模式,发现在卵产后36 h EsHE表达量开始大幅上升,至孵化前的48 h达到最高水平;且在3龄幼虫中肠组织高水平表达。这些结果暗示EsHE与家蚕追寄蝇的胚胎发育和孵化密切相关,还可能参与幼虫期的食物消化、蛋白质代谢等生物学过程。通过以家蚕追寄蝇基因组DNA为模板扩增发现,EsHE基因含有1个内含子(2 638 bp)和2个外显子(第1外显子1 005 bp,第2外显子502 bp),基因结构与铜绿蝇、丝光绿蝇HE相似。上述研究结果为进一步阐明家蚕追寄蝇孵化分子机理奠定了基础,为以孵化相关基因作为靶点的杀虫剂研究开发提供了理论基础。

Exorista sorbillans Wiedemann parasitizes silkworm larva and causes silkworm myiasis, which harms sericulture production. In order to study the hatching mechanism of E. sorbillans at the molecular level, the full-length cDNA of the hatching enzyme gene of E. sorbillans was obtained using 1.5-day-old fly eggs combined with RACE technology, which was named EsHE(GenBank Acc No.: ON042475). The full-length cDNA of EsHE is 1 507 bp, containing 159 bp 5 ′UTR, 412 bp 3′ UTR, and 936 bp ORF, encoding 311 amino acids. EsHE protein sequence contains HELMHVLGFMHE zinc-binding motif and YDYGSVMHY Met-turn motif, which are characteristic sequence of hatching enzyme. The phylogenetic tree analysis showed that the hatching enzymes of E. sorbillans and Lucilia sericata were most closely related. The expression pattern of EsHE gene was detected by qRT-PCR at the egg stage of E. sorbillans, and it was found that the expression level of EsHE began to increase sharply at 36 h after egg laying, and reached the highest level at 48 h before hatching. At the same time, the EsHE gene was expressed at a high level in the midgut tissue of the third instar larvae. These results suggest that EsHE is closely related to the embryonic development and hatching of E. sorbillans, and may also be involved in biological processes such as food digestion and protein metabolism in the larva stage. The genomic DNA of E. sorbillans was used as a template for amplification. The gene structure of EsHE contains one intron(2 638 bp) and two exons(1 005 bp in exon 1 and 502 bp in exon 2), which is similar to that of L. sericata and L. cuprina. The current results present a basis for further elucidating the molecular mechanism of the hatching process of E. sorbillans and developing a new pesticide using the hatching enzyme as target for silkworm myiasis.