黏虫丝氨酸蛋白酶抑制剂基因的克隆及抗球孢白僵菌功能分析

Cloning of the serine protease inhibitor from oriental armyworm Mythimna separata and analysis of its anti-Beauveria bassiana function

为探究丝氨酸蛋白酶抑制剂(serine protease inhibitor,Serpin)基因在黏虫Mythimna separata生长发育及免疫防御等过程中发挥的作用,在黏虫转录组中获得Serpin基因的cDNA序列,采用实时荧光定量PCR技术分析Serpin在黏虫不同发育阶段和不同组织中的表达水平,并通过RNA干扰技术分析Serpin在黏虫抗球孢白僵菌Beauveria bassiana过程中发挥的作用。结果显示,获得2个黏虫Serpin基因,命名为MsSerpin-4和Mserpin-5,其cDNA全长序列分别为1938 bp和2618 bp,开放阅读框分别为1188 bp和1197 bp,分别编码395个和398个氨基酸,GenBank登录号分别为MZ577062和MZ577063。2个基因在黏虫不同龄期和不同组织内均有表达,在成虫期的相对表达量最高,在1龄幼虫期的相对表达量最低;在中肠中的相对表达量最高,在唾腺中的相对表达量最低。RNA干扰6 h时对MsSerpin-4和Mserpin-5表达的抑制效果最好,抑制率分别为52.57%和49.15%;MsSerpin-4和Mserpin-5的表达被抑制后,黏虫对球孢白僵菌更敏感,校正死亡率分别提高了31.47%和14.62%。表明黏虫2个MsSerpin均参与了被球孢白僵菌侵染后的免疫反应,可为后续黏虫的生物防治提供新靶标。

To investigate the function of serine protease inhibitor(Serpin)in the growth,development,and immune defense of M.separata,the cDNA sequences of MsSerpin-4 and MsSerpin-5 were cloned from oriental armyworm Mythimna separata,and real-time quantitative PCR(qRT-PCR)was used to determine the expression levels of MsSerpins in various tissues and developmental stages.The RNA interference approach was employed to examine the function of MsSerpins in the anti-B.bassiana process.MsSerpin-4 and MsSerpin-5(GenBank accession numbers:MZ577062 and MZ577063)had sequence lengths of 1938 bp and 2618 bp,respectively,and open reading frames of 1188 bp and 1197 bp,encoding 395 and 398 amino acids,respectively.Both MsSerpins were expressed in different tissues and developmental stages.The first-instar larvae exhibited the lowest expression,while the adult stage showed the highest.Expression levels were the lowest in the salivary glands and highest in the midgut.The greatest inhibition of MsSerpins was achieved by RNA interference for six hours,resulting in 52.57% and 49.15% inhibition,respectively.Following MsSerpins inhibition,M.separata became more susceptible to B.bassiana,resulting in a 31.47% and 14.62% increase in corrected mortality,respectively.These findings suggest the involvement of two MsSerpins in the immunological response to B.bassiana infection,offering a new insight into the biological regulation of M.separata.