CAJ | 학술논문

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14～80 kD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.
위료획취여가잠황혈기인(Y)협동작용적상관단백적기출신식,통과쌍향전영기술대가잠황혈근등기인계5령기잠황혈개체화백혈개체적중장단백진행분리병작도상분석,발현분리출적교위청석적단백점주요집중재14～80 kD구역,등전점(pI) 4～9.채용기질보조격광해석전리비행시간질보(MALDI-TOF-MS)기술대기중일사차이명현적단백점진행감정,감정결과교위가신적5개단백점포괄삼여능량대사、단백질합성등공능단백,저사단백가능여가잠황혈성상적형성유관.채용실시정량PCR방법,대질보감정출적질자전이ATP합매β아기2편마기인재가잠황혈근등기인계황혈개체화백혈개체5령불동발육시기적상대표체량진행분석,결과표명해기인재2충개체중적표체균정현명현상승적추세,병차재황혈개체중적표체량명현교백혈개체고,5령제1-2천적상대표체량고7배좌우,제시해기인가능여가잠적황혈성상유관련.
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).Dis- tinct 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 i-dentifying 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 expres-sion levels of H+ transposing 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+ trans-porting ATP synthase β subunit isoform 2 gene wereup-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.