家蚕分泌型磷脂酶A2基因的鉴定和表达模式分析

Identification and expression pattern analysis of a secretory phospholipase A2 gene in Bombyx mori

磷脂酶A2(phospholipase A2,PLA2)广泛分布于动物、植物和微生物,参与许多生理活动。本实验室先前鉴定和研究了1个家蚕分泌型PLA2(Bombyx mori sPLA2-1-1)基因功能。最近,从家蚕基因组鉴定到4个新的sPLA2基因(BmsPLA2-1-2、BmsPLA2-2、BmsPLA2-3、BmsPLA2-4),预测的氨基酸都具有sPLA2超家族结构域、金属离子结合位点和高度保守的催化位点等sPLA2的典型特征。本研究完成了BmsPLA2-4基因的克隆、体外表达和表达模式分析。BmsPLA2-4全长为585 bp,体外表达经Ni纯化获得重组蛋白大小约25 kDa。qRT-PCR分析表明,BmsPLA2-4在5龄第1天的表达量最高。组织表达谱分析表明,BmsPLA2-4在中肠的表达量最高,其次是体壁和脂肪体。Western blotting分析结果与qRT-PCR一致。用大肠杆菌和金黄色葡萄球菌感染5龄第1天幼虫后,BmsPLA2-4基因的表达量在24 h显著升高。本研究为后续研究提供了理论基础和有价值的实验数据。

Phospholipase A2(PLA2)is widely distributed in animals,plants,and microorganisms,and it plays an important role in many physiological activities.In a previous study,we have identified a secretory PLA2 in Bombyx mori(BmsPLA2-1-1).In this study,we further identified four new sPLA2 genes(BmsPLA2-1-2,BmsPLA2-2,BmsPLA2-3,and BmsPLA2-4)in B.mori genome.All four genes exhibits the characteristic features of sPLA2,including the sPLA2 domain,metal binding sites,and highly conserved catalytic domain.This study completed the cloning,in vitro expression,and expression pattern analysis of the BmsPLA2-4 gene in B.mori.The full length of BmsPLA2-4 is 585 bp,and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa.qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage.Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut,followed by the epidermis and fat body.Western blotting analysis results were consistent with those of qRT-PCR.Furthermore,after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus,the expression level of the BmsPLA2-4 gene significantly increased in 24 h.The findings of this study provides a theoretical basis and valuable experimental data for future related research.