短时热激后家蚕对核型多角体病毒的抗性及相关抗菌肽和热激蛋白基因表达水平的变化

Changes in the resistance to nucleopolyhedrovirus and the expression levels of the related antimicrobial peptide and heat shock protein genes in Bombyx mori exposed to short-time heat shock

【目的】探讨短时热激对家蚕Bombyx mori抵抗核型多角体病毒能力的影响,为深入研究家蚕热激响应的抗病毒免疫机制提供参考。【方法】家蚕5龄幼虫42℃热激15 min后,经口喂食家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,Bm NPV)(5.4×106PIBs/头)后正常饲养,观察热激对家蚕感染病毒的影响;同时,利用实时荧光定量RT-PCR技术检测热激后7类抗菌肽主基因(Bmcec D,Bmmor,Bmglve2,Bmdefe B,Bmatta1,Bmenbo2和Bmlebo3)以及6种热激蛋白基因(Bmhsp90,Bmhsp70,Bmhsp40,Bmhsp19.9,Bmhsp21.4和Bmhsp25.4)在家蚕5龄幼虫中肠组织中的表达变化;利用MEGA 5.0软件对家蚕所有热激蛋白进行系统进化分析。【结果】与未进行热激处理相比,短时42℃热激能够明显提高感染Bm NPV的家蚕5龄幼虫的存活率。实时荧光定量RT-PCR结果表明,与未热激处理相比,热激处理后感染Bm NPV的家蚕5龄幼虫中肠组织中Bmglve2和Bmhsp25.4的表达量显著上升。另外,系统进化分析显示,小分子热激蛋白Bm HSP25.4在进化上特殊,单独与大分子热激蛋白(Bm HSP90,Bm HSP70和Bm HSP40)聚为一类。【结论】短时热激能够提高家蚕对Bm NPV的抗性,热激蛋白基因Bmhsp25.4和抗菌肽基因Bmglve2可能在家蚕热激响应的抗病毒免疫中发挥关联功能。

【Aim】This study aims to investigate the effect of short-time heat shock on the resistance of the silkworm,Bombyx mori to nucleopolyhedrovirus,so as to provide references for further study on the antiviral immune mechanism of the silkworm in response to heat shock.【Methods】The 5th instar larvae of B.mori were exposed to 42℃ for 15 min,and then fed with B.mori nucleopolyhedrovirus（Bm NPV）（5.4 × 106PIBs/individual）.All the tested larvae were reared as usual.The transcription levels of seven major antimicrobial genes（Bmcec D,Bmmor,Bmglve2,Bmdefe B,Bmatta1,Bmenbo2 and Bmlebo3）and six heat shock protein genes（Bmhsp90,Bmhsp70,Bmhsp40,Bmhsp19.9,Bmhsp21.4 and Bmhsp25.4） in the midgut of the 5th instar larvae after heat-shock treatment were analyzed by quantitative real-time PCR.The amino acid sequences of all Bm HSPs were also phylogenetically analyzedusing MEGA 5.0.【Results 】 Short-time heat shock significantly increased the survival rate of the Bm NPV-infected 5th instar larvae of B.mori,as compared with the non-heat shock group.Quantitative real-time PCR results showed that the expression levels of Bmglve2 and Bmhsp25.4 were significantly upregulated in the midgut of the Bm NPV-infected 5th instar larvae of B.mori exposed to heat shock in comparison with the non-heat shock groups.The phylogenetic analysis showed that Bm HSP25.4 was special in evolution,and individually classified into the same cluster with large HSPs（Bm HSP90,Bm HSP70 and Bm HSP40）.【Conclusion】Short-time heat shock before Bm NPV feeding can enhance the resistance of B.mori to Bm NPV.Bmhsp25.4 and Bmglve2 may relatively function in the immunity induction of B.mori against Bm NPV through heat shock response.