激素和限食处理对家蚕SPs与Serpins基因表达的影响
The expression of serine proteinases (SPs) and serine protease inhibitor (Serpins) genes in silkworms, Bombyx mori, treated with hormones and subject to food restriction
甘丽萍1** 刘仁华1 龙菲菲2 张 进2 徐世清2
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DOI：10.7679/j.issn.2095-1353.2014.149
作者单位：1. 重庆三峡学院 生命科学学院，万州 404000；2. 苏州大学 现代丝绸国家工程实验室，苏州 215123
中文关键词：家蚕，丝氨酸蛋白酶，丝氨酸蛋白酶抑制剂，激素和限食处理，基因表达
英文关键词：Bombyx mori, serine proteinases (SPs), Serine protease inhibitor (Serpins), hormone and food restriction treatment, gene expression
中文摘要： 【目的】 为了调查激素和限食处理对家蚕幼虫丝氨酸蛋白酶（SPs）与丝氨酸蛋白酶抑制剂（Serpins）基因表达的影响。【方法】 使用RT-PCR、实时定量PCR以及Western-blot的方法研究了部分SPs、Serpins基因在mRNA和蛋白质水平上的表达情况，以及20-羟基蜕皮酮（20-hydroxyecdysone，20E）、保幼激素类似物（JHA）和限食处理后SPs与Serpins家族基因的变化。【结果】 调查的基因在mRNA和蛋白质水平上的表达情况一致，SPs基因在中肠中特异表达，5龄中期达到顶峰，而Serpins基因主要在丝腺中表达，5龄中后期的表达较高；20E处理5龄3日家蚕幼虫，SPs和Serpins基因呈现不同的调控趋势，JHA对这两类基因的表达具有类似的正调控作用，激素对基因的调控主要与发育时期及激素剂量有关；Spi1基因在幼虫饥饿处理后出现下调比Spp晚，暗示Serpins受取食的直接影响比SPs慢，也说明了食物供需对丝腺的反应迟于对中肠的影响。SPs和Serpins基因的这种组织表达特异性，为它们参与相应组织中的特异性转录调控提供了佐证，而发育时期表达差异和激素及限食处理后调控变化体现了基因执行功能上的时序性和特殊性。【结论】 家蚕丝物质合成效率与SPs 及Serpins基因在mRNA和蛋白水平上的表达有关，这将有助于更好地理解家蚕丝物质形成和积累的调控机理。
英文摘要：
[Objectives] To investigate the effects of hormone treatment and food restriction on the gene expression of serine proteases (SPs) and serine protease inhibitors (Serpins) in Bombyx mori. [Methods] mRNA expression and protein levels of SPs and Serpin genes were tested by RT-PCR，Real time PCR and western blot，in silkworms treated with 20-hydroxyecdysone (20E), juvenile (JHA) hormones and food restriction. [Results] mRNA expression was consistent with protein levels. SPs were especially expressed in the midgut of mid-fifth instar, however Serpins were mainly expressed in the silk glands, achieving a higher level in the late fifth instar. SPs and Serpin genes displayed different regulatory trends in the fifth and third instar silkworm larvae treated with 20E and JHA. Hormone regulatory effect was mainly influenced by developmental stages and dosage. The expression of the Spi1 gene was down regulated after food restriction, in addition this process occurred later than for the Spp gene, which implies that food restriction directly affects Serpins. It also illustrates that the silk glands respond faster to food supply than the midgut. The tissue specificity of SPs and Serpins demonstrates that they participate in tissue-specific transcriptional regulation. Both SPs and Serpins were differentially expressed in different developmental stages in response to either hormone treatment or food restriction. [Conclusion] These findings on the function and specificity of SP and Serpins further understanding of the mechanism of silk formation and accumulation.