摘要
采用银染mRNA差异显示方法从绍鸭卵巢等级卵泡中分离并筛选到 3个差异表达序列标签 (ExpressedSequenceTags ,ESTs)SXDF0 2 0 1(2 71bp)、SXDF0 2 0 2 (2 0 0bp)和SXDF0 2 0 3(173bp) ,通过测序和BLAST检索 ,发现SXDF0 2 0 1与GenBank中登录的所有物种的所有序列均无同源性 ,是在绍鸭卵巢卵泡发现的新EST ,现已登录GenBank(GenBank登录号 :CB0 72 6 2 9) ,而SXDF0 2 0 2和SXDF0 2 0 3则分别与GenBank公布的鸡的EST和肌胃肌球蛋白重链高度同源。用 5′ RACE将SXDF0 2 0 1延伸至 5 4 4bp ,经过BLAST再次检索 ,仍然未发现中度同源序列采用相对定量RT PCR方法进一步研究SXDF0 2 0 1和SXDF0 2 0 2在绍鸭组织中的时空特异性表达 ,发现这两个EST在产蛋高峰期绍兴鸭的下丘脑、垂体、肌肉、肝脏、脂肪等组织都有表达 ;SXDF0 2 0 1在 30日龄卵巢的表达水平显著高于 6 0 (P <0 0 5 )和 90 (P =0 0 15 )日龄 ;而SXDF0 2 0 2在卵巢发育的不同阶段的表达水平没有变化 ;SXDF0 2 0 1在卵巢卵泡颗粒层中的表达总体高于膜层 ,SXDF0 2 0 2在颗粒层中的表达F3 >F5>Fw(P <0 0 1) ,而在F1卵泡降至最低 (P <0 0 1) ,膜层中以Fw 卵泡表达水平最高 (P <0 0 1)。
Three expressed sequence tags (ESTs),SXDF0201(271 bp),SXDF0202 (200 bp) and SXDF0203 (173 bp),were isolated from ovarian follicles of Shaoxing ducks by using silver staining mRNA differential display.GenBank/BLAST analysis revealed that SXDF0201 was not homologous to any of the published sequences from all species,indicating that it was a novel EST and was then registered in GenBank (GenBank Accession No.: CB072629),while SXDF0202 and SXDF0203 were found to be highly homologous to seven known chicken ESTs and chicken mRNA for gizzard smooth muscle myosin heavy chain.5′-RACE was employed to extend the SXDF0201 to 544 bp which was confirmed as novel in BLAST search.The temporal and spatial expression of SXDF0201 and SXDF0202 were also investigated with semi-quantitative RT-PCR.The result showed that: both SXDF0201 and SXDF0202 were found to be expressed in hypothalamus,pituitary,muscle,liver,and fat tissues of Shaoxing ducks; SXDF0201 was expressed significantly higher in ovaries of 30-day-old Shaoxing ducks compared with that of 60-day-old (P<0.05) and 90-day-old (P=0.015),but the expression of SXDF0202 showed no difference throughout the ovarian development; granulose layers expressed higher SXDF0201 than theca layers in almost all hierarchical follicles,the expression of SXDF0202 in granulose layers increased along with follicular maturation (P<0.01) from F_w to F_3 follicles,but decreased dramatically to the lowest in F_1 follicles (P<0.01).In theca layers,the highest expression of SXDF0202 was found in F_w follicles (P<0.01).
基金
国家自然科学基金农业倾斜项目 (编号 :3 9770 5 40 )资助~~
关键词
差异显示
绍兴鸭
卵巢卵泡
表达序列标签
silver staining mRNA differential display
Shaoxing duck
ovarian follicles
expressed sequence tags
