饥饿胁迫下家蚕5龄起蚕中的蛋白表达谱及BmLp-c6的转录分析

Protein expression profiles and the transcriptional analysis of Bm Lp-c6 in the 5th instar larvae of the silkworm( Bombyx mori) under starvation stress

[目的]分析家蚕Bombyx mori受饥饿胁迫后的蛋白质谱变化,探索其耐受饥饿的机理。[方法]以家蚕品种932为实验材料,利用双向电泳和质谱技术检测5龄起蚕经过24 h饥饿胁迫的蛋白质谱差异变化,利用荧光定量PCR技术分析Bm Lp-c6的转录表达。[结果]经比对饥饿蚕和正常取食蚕的血淋巴蛋白谱,饥饿蚕有62个特异蛋白点。蛋白点的等电点在4.22～6.98之间,分子量分布在20.81～144.69 k Da间。选取只在饥饿时出现的特异蛋白点No.7111进行质谱鉴定,根据其氨基酸序列进行引物设计,获得了目的基因Bm Lp-c6,经与载体p ET-21d（＋）连接重组后,成功获得诱导表达。经实时荧光定量PCR分析,当5龄起蚕受到饥饿胁迫影响时,Bm Lp-c6基因在血淋巴中大量转录表达,但在中肠中的转录表达水平却极低。[结论]家蚕5龄起蚕在饥饿胁迫下,血淋巴中的蛋白质谱发生变化,Bm Lp-c6会大量转录表达。

[Aim]This study aims to analyze the changes in protein expression in the silkworm,Bombyx mori,under starvation stress,so as to illustrate the mechanism of starvation tolerance of the silkworm.[Methods]The silkworm variety 932 was used as the experimental material. The difference of protein expression was analyzed through two-dimensional electrophoresis and mass spectrometry technology between the silkworms normally feeding and subjected to 24 h starvation stress after the 5th instar newly moulting larval stage. The transcription level of Bm Lp-c6 was detected by using real-time quantitative PCR. [Results]There were 62 specific proteins in the total proteins of the starved larvae compared with the larvae normally feeding. Their isoelectric points range from 4. 22 to 6. 98,and their molecular weights are distributed between 20. 81 and 144. 69 k Da. The specific protein No. 7111,which was expressed only in the starved larvae,was selected and identified by mass spectrometry. According to the amino acid sequence,the primers were designed and the target gene Bm Lp-c6 was cloned. The Bm Lp-c6 gene was induced to express successfully after being connected with the expression vector p ET-21d（ ＋）. The realtime quantitative PCR analysis showed that when the 5th instar larvae suffered from starvation stress,Bm Lp-c6 had a higher transcriptional level in the hemolymph,while its transcriptional level was very low in the midgut. [Conclusion]When the 5th instar larvae of B. mori are subjected to starvation stress,the protein expression profiles in the hemolymph change and Bm Lp-c6 has a high transcriptional level.