CAJ | 학술논문

已知多数含BTB/POZ结构域的锌指蛋白对生物体的发育、分化及染色体重组等具有重要的调节作用.从已构建的家蚕蛹cDNA文库中筛选到一条编码MOD(mdg4)蛋白的基因序列(GenBank登录号:DN237077).该基因序列的ORF长1 080 bp,编码359个氨基酸残基,蛋白含有BTB保守结构域和FLYWCH保守结构域,与黑腹果蝇、冈比亚按蚊、赤拟谷盗和埃及伊蚊的MOD多序列比对,其同源性不超过40%.表达、纯化带有His标签的家蚕MOD蛋白(BmMOD),并用纯化的融合蛋白免疫新西兰兔获得多克隆抗体.实时荧光定量PCR显示BmMod基因mRNA转录水平在家蚕蛾期及5龄幼虫的卵巢最高,推测BmMod基因在家蚕生殖腺发育过程中产生一定作用.利用纯化的BmMOD蛋白多克隆抗体进行Western blotting分析也表明该蛋白在家蚕蛾期及5龄幼虫卵巢中的表达量最高,验证了BmMod在家蚕生殖腺发育中的功能.亚细胞定位结果显示BmMOD蛋白几乎完全定位于家蚕卵巢上皮细胞(BmN)的细胞核中.
이지다수함BTB/POZ결구역적자지단백대생물체적발육、분화급염색체중조등구유중요적조절작용.종이구건적가잠용cDNA문고중사선도일조편마MOD(mdg4)단백적기인서렬(GenBank등록호:DN237077).해기인서렬적ORF장1 080 bp,편마359개안기산잔기,단백함유BTB보수결구역화FLYWCH보수결구역,여흑복과승、강비아안문、적의곡도화애급이문적MOD다서렬비대,기동원성불초과40%.표체、순화대유His표첨적가잠MOD단백(BmMOD),병용순화적융합단백면역신서란토획득다극륭항체.실시형광정량PCR현시BmMod기인mRNA전록수평재가잠아기급5령유충적란소최고,추측BmMod기인재가잠생식선발육과정중산생일정작용.이용순화적BmMOD단백다극륭항체진행Western blotting분석야표명해단백재가잠아기급5령유충란소중적표체량최고,험증료BmMod재가잠생식선발육중적공능.아세포정위결과현시BmMOD단백궤호완전정위우가잠란소상피세포(BmN)적세포핵중.
Most BTB/POZ-domain-containing zinc finger proteins play important regulatory roles in development, differentiation and chromatin remodeling etc. A cDNA sequence which encodes Mod(mdg4) protein was obtained in a previously constructed cDNA library for silkworm pupae (GenBank accession number DN237077). It has an open reading frame of 1 080 bp which encodes a polypeptide of 359 amino acids. This protein has two conserved domains: a BTB domain and an FLYWCH motif. Its sequence identity with Drosophila melanogaster,Tribolium castaneum,Anopheles gambiae,and Aedes aegypti was all below 40%. The His-tagged silkworm MOD protein (BmMOD) was expressed in E.coli BL21 strain and was subsequently purified. The purified fusion protein was used to immune New Zealand rabbit for preparing polyclonal antibodies. Real time fluorescent quantitative PCR revealed that transcriptional levels of BmMOD mRNA were the highest in ovary of the 5th instar larva and in moth,suggesting that BmMOD gene is involved in silkworm gonad development. Moreover, Western blotting analysis using polyclonal antibodies against the purified BmMOD protein indicated that the expression amount of BmMOD protein was the highest in silkworm moth and in the ovary of the 5th instar larva,providing further evidence for its involvement in silkworm gonad development. Subcellular localization result showed that almost all BmMOD proteins were located in the nuclei of the silkworm ovarian epithelial (BmN) cells.