摘要
为了获取与家蚕黄血基因(Y)协同作用的相关蛋白的基础信息,通过双向电泳技术对家蚕黄血近等基因系5龄起蚕黄血个体和白血个体的中肠蛋白进行分离并作图像分析,发现分离出的较为清晰的蛋白点主要集中在14～80kD区域,等电点(pI)4～9。采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术对其中一些差异明显的蛋白点进行鉴定,鉴定结果较为可信的5个蛋白点包括参与能量代谢、蛋白质合成等功能蛋白,这些蛋白可能与家蚕黄血性状的形成有关。采用实时定量PCR方法,对质谱鉴定出的质子转移ATP合酶β亚基2编码基因在家蚕黄血近等基因系黄血个体和白血个体5龄不同发育时期的相对表达量进行分析,结果表明该基因在2种个体中的表达均呈现明显上升的趋势,并且在黄血个体中的表达量明显较白血个体高,5龄第1-2天的相对表达量高7倍左右,提示该基因可能与家蚕的黄血性状有关联。
In order to acquire basic information of related proteins which function synergistically with silkworm （Bombyx mori） yellow hemolymph gene （Y）, we employed two-dimensional polyacrylamide gel electrophoresis and ImageMaster 2-D platinum approach to separate and analyze midgut proteins of the 5th instar newly exuviated silkworm larvae with yellow and white hemolymph respectively of a yellow hemolymph near-isogenic line （NIL）. Distinct protein spots on silver-stained gel were mainly distributed in an area from 14 to 80 kD with pl from 4 to 9. The matrix-assisted laser desorption ionization and time of flight mass spectrometry （MALDI-TOF-MS） was applied for identifying distinct protein spots on the 2-D map. 5 spots were identified with relatively high reliability to involve in many biological processes including energy metabolism and protein synthesis. These proteins are probably related to the formation of silkworm yellow hemolymph character. Real-time quantitative PCR analysis to the relative expression levels of H ＋ transporting ATP synthase β subunit isoform 2 gene in different developmental stages of the NIL＇ s 5th instar individuals with yellow and white hemolymph respectively revealed that the expression levels of H ＋ transporting ATP synthase β subunit isoform 2 gene were up-regulated obviously in both individuals. Nevertheless, the expression level in individuals with yellow hemolymph was remarkably higher than that with white hemolymph, being 7-fold higher in day 1 to day 2, suggesting a close relationship between this gene and the yellow hemolymph phenotype in silkworm.
出处
《蚕业科学》
CAS
CSCD
北大核心
2010年第2期250-255,共6页
ACTA SERICOLOGICA SINICA
基金
现代农业产业技术体系建设专项(蚕桑)
重点基础研究发展计划"973"项目(No.2005CB121004)
国家科技支撑计划项目(No.2007BAD72B01)
江苏省科技项目(No.BG2007322)
关键词
家蚕
黄血近等基因系
双向电泳
质谱分析
实时定量PCR
Bombyx mori Near-isogenic line（NIL） Two-dimensional polyacrylamide gel electrophoresis Mass spec-trometry Real-time quantitative PCR
