Cao Hui-Hua, Zhang Shang-Zhi, Zhu Lin-Bao, Wang Jie, Liu Ying-Xue, Wang Yu-Ling, Kong Xue, You Ling-Ling, Toufeeq Shahzad, Liu Shi-Huo, Xu Jia-Ping
School of Life Sciences, Anhui Agricultural University, Hefei, 230036, China; Anhui International Joint Research and Developmental Center of Sericulture Resources Utilization, Hefei, 230036, China.
Hunan Provincial Collaborative Innovation Center for Field Weeds Control, Hunan University of Humanities, Science and Technology, Loudi, 417000, China.
Dev Comp Immunol. 2021 Jun;119:104035. doi: 10.1016/j.dci.2021.104035. Epub 2021 Jan 31.
Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious pathogenic microorganism that causes tremendous loss to sericulture. Previous studies have found that some proteins of serine protease family in the digestive juice of B. mori larvae have anti-BmNPV activity. In our previous publication about proteome analysis of the digestive juice of B. mori larvae, the digestive enzyme trypsin, alkaline A (BmTA) was filtered as a differentially expressed protein possibly involved in BmNPV resistance. Here, the biological characteristics and anti-BmNPV functions of BmTA were comprehensively analysed. The cDNA sequence of BmTA had an ORF of 768 nucleotides encoding 255 amino acid residues. Domain architecture analysis showed that BmTA contained a signal peptide and a typical Tryp_SPc domain. Quantitative real-time PCR analysis showed that BmTA was highly expressed in the larval stages and specifically expressed in the midgut of B. mori larvae. The expression level of BmTA in BmNPV resistant strain A35 was higher than that in susceptible strain P50. After BmNPV infection, the expression of BmTA increased in both strains from 24 to 72 h. Virus amplification analysis showed that the relative levels of VP39 in B. mori larvae and BmN cells infected with the appropriate concentration of recombinant-BmTA-treated BmNPV were significantly lower than in the control groups. Moreover, overexpression of BmTA in BmN cells significantly inhibited the amplification of BmNPV. Taken together, the results of this study indicated that BmTA possessed anti-BmNPV activity in B. mori, which broadens the horizon for virus-resistant breeding of silkworms.
家蚕核型多角体病毒（BmNPV）是一种严重的病原微生物，给养蚕业造成巨大损失。先前的研究发现，家蚕幼虫消化液中丝氨酸蛋白酶家族的一些蛋白质具有抗BmNPV活性。在我们之前关于家蚕幼虫消化液蛋白质组分析的出版物中，消化酶胰蛋白酶碱性A（BmTA）作为可能参与BmNPV抗性的差异表达蛋白被筛选出来。在此，对BmTA的生物学特性和抗BmNPV功能进行了全面分析。BmTA的cDNA序列有一个768个核苷酸的开放阅读框，编码255个氨基酸残基。结构域分析表明，BmTA包含一个信号肽和一个典型的Tryp_SPc结构域。定量实时PCR分析表明，BmTA在幼虫阶段高表达，在家蚕幼虫中肠特异性表达。BmTA在BmNPV抗性品系A35中的表达水平高于敏感品系P50。BmNPV感染后，两个品系中BmTA的表达在24至72小时内均增加。病毒扩增分析表明，用适当浓度的重组BmTA处理的BmNPV感染的家蚕幼虫和BmN细胞中VP39的相对水平显著低于对照组。此外，BmTA在BmN细胞中的过表达显著抑制了BmNPV的扩增。综上所述，本研究结果表明BmTA在家蚕中具有抗BmNPV活性，这为家蚕抗病毒育种拓宽了视野。
