DataSheet_1_BmSPP is a virus resistance gene in Bombyx mori.docx

IntroductionSignal peptide peptidase (SPP) is an intramembrane protease involved in a variety of biological processes, it participates in the processing of signal peptides after the release of the nascent protein to regulate the endoplasmic reticulum associated degradation (ERAD) pathway, binds misfolded membrane proteins, and aids in their clearance process. Additionally, it regulates normal immune surveillance and assists in the processing of viral proteins. Although SPP is essential for many viral infections, its role in silkworms remains unclear. Studying its role in the silkworm, Bombyx mori , may be helpful in breeding virus-resistant silkworms.
MethodsFirst, we performed RT-qPCR to analyze the expression pattern of BmSPP. Subsequently, we inhibited BmSPP using the SPP inhibitor 1,3-di-(N-carboxybenzoyl-L-leucyl-L-leucylaminopropanone ((Z-LL)2-ketone) and downregulated the expression of BmSPP using CRISPR/Cas9 gene editing. Furthermore, we assessed the impact of these interventions on the proliferation of Bombyx mori nucleopolyhedrovirus (BmNPV).
ResultsWe observed a decreased in the expression of BmSPP during viral proliferation. It was found that higher concentration of the inhibitor resulted in greater inhibition of BmNPV proliferation. The down-regulation of BmSPP in both in vivo and in vitro was found to affect the proliferation of BmNPV. In comparison to wild type silkworm, BmSPPKO silkworms exhibited a 12.4% reduction in mortality rate.
DiscussionCollectively, this work demonstrates that BmSPP plays a negative regulatory role in silkworm resistance to BmNPV infection and is involved in virus proliferation and replication processes. This finding suggests that BmSPP servers as a target gene for BmNPV virus resistance in silkworms and can be utilized in resistance breeding programs.

引言
信号肽肽酶（signal peptide peptidase, SPP）是一类参与多种生物学过程的膜内蛋白酶。该酶可在新生蛋白质释放后参与信号肽的加工过程，以调控内质网相关降解（endoplasmic reticulum associated degradation, ERAD）通路，结合错误折叠的膜蛋白并协助其清除过程。此外，SPP还可调控正常免疫监视，并参与病毒蛋白的加工。尽管SPP对多种病毒感染至关重要，但其在家蚕（Bombyx mori）中的作用仍不明确。探究其在家蚕中的功能，有助于培育抗病毒家蚕品种。
方法
首先，我们通过实时定量聚合酶链反应（real-time quantitative polymerase chain reaction, RT-qPCR）分析BmSPP的表达模式。随后，我们使用SPP抑制剂1,3-二-(N-羧基苯甲酰基-L-亮氨酰-L-亮氨酰氨基丙酮)（(Z-LL)2-ketone）抑制BmSPP的活性，并通过CRISPR/Cas9基因编辑技术下调BmSPP的表达。此外，我们评估了这两种干预手段对家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus, BmNPV）增殖的影响。
结果
我们观察到病毒增殖过程中BmSPP的表达水平下调。研究发现，抑制剂浓度越高，对BmNPV增殖的抑制效果越强。体内与体外实验均显示，BmSPP的下调会影响BmNPV的增殖。与野生型家蚕相比，BmSPP基因敲除（BmSPPKO）家蚕的死亡率降低了12.4%。
讨论
本研究整体证明，BmSPP在家蚕抗BmNPV感染过程中发挥负调控作用，并参与病毒的增殖与复制过程。这一发现表明，BmSPP可作为家蚕抗BmNPV病毒的靶基因，应用于抗病育种项目中。