摘要
柞蚕是一种典型的野蚕,其丝腺能够合成和分泌丝蛋白。在五龄期,柞蚕幼虫大量合成和分泌丝素蛋白,后部丝腺的亚细胞结构和新陈代谢也发生了很大的变化。为研究差异表达蛋白在丝素蛋白准备和合成过程中所起的作用,本论文通过双向电泳和质谱的方法分离和鉴定了五龄柞蚕幼虫丝腺中表达量上调的蛋白,并通过实时定量PCR检测了其中两个蛋白的RNA表达变化。
样品的准备是双向凝胶电泳鉴定成功与否的关键一步。为得到较高分辨率的蛋白电泳图谱,实验通过优化,选用两步提取法提取柞蚕丝腺组织蛋白,将水溶性蛋白和微溶或不溶的蛋白分开,防止高丰度蛋白对双向电泳的影响;并用2D clean-up试剂盒对蛋白溶液进行了预处理,减少了横条纹。
实验采用载体两性电解质进行等点聚焦,建立了一套简单经济的双向电泳方法,并对柞蚕丝腺蛋白双向电泳的条件进行了优化,得到此系统的最佳上样量为:30μg;最佳pH范围:pH4～7;最佳聚焦条件:1000V,3h。通过和五龄一天柞蚕丝腺蛋白的双向电泳图谱相比较发现:在五龄四天时蛋白表达有较大的差异。
采用IPG固化胶条进行双向电泳,得到了分辨率较高的五龄一天和四天柞蚕丝腺蛋白双向电泳图谱,每张图谱检测到大约350个蛋白点。通过Imagemaster软件进行比对分析,选取了23个在五龄四天特异表达或表达量上调两倍以上的蛋白进行MALDI-TOF-MS鉴定,得到了7个已知蛋白,这些蛋白在转录、翻译和细胞新陈代谢中起着重要的作用。实验对其中的两个蛋白基因(谷胱甘肽硫转移酶和核糖体蛋白L8)进行了克隆,获得了Genbank登录号为EU541490和EU541491。另外,通过实时定量PCR对这两个蛋白进行了RNA转录水平分析,结果发现,RNA表达水平和蛋白表达水平相一致。
Antheraea pernyi is a typical wild silkworm with larval silk glands specific for the synthesis and secretion of silk proteins.When the larva develops to the fifth instar stage, fibroin is synthesized and secreted in abundance and the metabolism and subcellular structure of the PSG undergoes considerable changes.To study the functions of different expression proteins associated with fibrion preparation and synthesis,two-dimensional electrophoresis and MALDI-TOF-MS were used to identify the upregulated proteins in PSG of A.pernyi fifth instar larvae.Real-time quantitative PCR was performed to detect changes of GSTT and RPL8 at RNA transcription level.
Sample preparation is the first and important step towards successful two-dimensional gel electrophoresis(2-DE) and identification in proteomics study.Two-step method which separated the soluble and disolube proteins was proved to be more suitable for silk glands protein extraction to avoid the influence of abundant protein on 2-DE.2D clean-up kit was used to provent horizontal streaks.
A simple and economic IEF was performed by Bio-lyte for 2-DE.The optimal condition for 2-DE of PSG protein was 30μg;pH 4～7;1000V,3h.By comparing the 2-DE profiles with day 1 of fifth instar,more changes of protein spots were found on day 4.
The 2-DE profiles of days 1 and 4 were obtained through the method of IPG strip and about 350 spots were detected on each profile.After analyzing by Imagemaster,23 unique or upregulated spots(above two folds) on day 4 of the fifth instar were selected to be identified by MALDI-TOF-MS.After searching NCBI database,seven proteins which probably involved in the regulation of transcription,translation,and general cell metabolism were identified.GSTT and RPL8 were cloned and its Genbank accession numbers were EU541490 and EU541491.Real-time quantitative PCR was performed on these two genes to determine the changes at RNA transcription level.The result revealed that the changes at RNA expression level were in corresponding with the protein expression level.
引文
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