Zheng Shiyi, Huang Yukang, Huang Hongyun, Yu Bin, Zhou Ni, Wei Junhong, Pan Guoqing, Li Chunfeng, Zhou Zeyang
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400715, China.
Chongqing Key Laboratory of Microsporidia Infection and Control, Southwest University, Chongqing, 400715, China.
Parasit Vectors. 2021 Jan 25;14(1):81. doi: 10.1186/s13071-021-04595-8.
Nosema bombycis is a unicellular eukaryotic pathogen of the silkworm, Bombyx mori, and is an economic and occupational hazard in the silkworm industry. Because of its long incubation period and horizontal and vertical transmission, it is subject to quarantine measures in sericulture production. The microsporidian life-cycle includes a dormant extracellular phase and intracellular proliferation phase, with the proliferation period being the most active period. This latter period lacks spore wall protection and may be the most susceptible stage for control.
In order to find suitable target for the selective breeding of N. bombycis-resistant silkworm strains, we screen highly expressed membrane proteins from the transcriptome data of N. bombycis. The subcellular localization of the candidate protein was verified by Indirect immunofluorescence analysis (IFA) and immunoelectron microscopy (IEM), and its role in N. bombycis proliferation was verified by RNAi.
The N. bombycis protein (NBO_76g0014) was identified as a transmembrane protein and named NbTMP1. It is homologous with hypothetical proteins NGRA_1734 from Nosema granulosis. NbTMP1 has a transmembrane region of 23 amino acids at the N-terminus. Indirect immunofluorescence analysis (IFA) results suggest that NbTMP1 is secreted on the plasma membrane as the spores develop. Western blot and qRT-PCR analysis showed that NbTMP1 was expressed in all developmental stages of N. bombycis in infected cells and in the silkworm midgut. Downregulation of NbTMP1 expression resulted in significant inhibition of N. bombycis proliferation.
We confirmed that NbTMP1 is a membrane protein of N. bombycis. Reduction of the transcription level of NbTMP1 significantly inhibited N. bombycis proliferation, and this protein may be a target for the selective breeding of N. bombycis-resistant silkworm strains.
家蚕微孢子虫是家蚕的一种单细胞真核病原体，对养蚕业构成经济和职业危害。由于其潜伏期长且具有水平和垂直传播特性，在养蚕生产中需采取检疫措施。微孢子虫的生命周期包括休眠的细胞外阶段和细胞内增殖阶段，增殖期是最活跃的时期。后一时期缺乏孢子壁保护，可能是控制的最敏感阶段。
为了找到抗家蚕微孢子虫品系选育的合适靶点，我们从家蚕微孢子虫的转录组数据中筛选高表达膜蛋白。通过间接免疫荧光分析（IFA）和免疫电子显微镜（IEM）验证候选蛋白的亚细胞定位，并通过RNA干扰验证其在家蚕微孢子虫增殖中的作用。
家蚕微孢子虫蛋白（NBO_76g0014）被鉴定为跨膜蛋白，并命名为NbTMP1。它与颗粒体微孢子虫的假定蛋白NGRA_1734同源。NbTMP1在N端有一个由23个氨基酸组成的跨膜区域。间接免疫荧光分析（IFA）结果表明，随着孢子的发育，NbTMP1分泌到质膜上。蛋白质免疫印迹和qRT-PCR分析表明，NbTMP1在家蚕微孢子虫感染细胞和家蚕中肠的所有发育阶段均有表达。下调NbTMP1的表达导致家蚕微孢子虫增殖受到显著抑制。
我们证实NbTMP1是家蚕微孢子虫的膜蛋白。降低NbTMP1的转录水平显著抑制了家蚕微孢子虫的增殖，该蛋白可能是抗家蚕微孢子虫品系选育的靶点。
