鸡原始生殖细胞中lncRNA-CEPT8编码小肽能力的研究
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摘要
为探究lncRNA在鸡生殖细胞中的调控作用,以如皋黄鸡受精蛋为试验材料,基于高通量测序筛选结果,利用RACE技术获取lncRNA-CEPT8序列全长,通过荧光定量检测和核质分离试验确定lncRNA-CEPT8的富集位置,并进行ORF开放阅读框预测及构建原核融合表达载体,提取蛋白质后利用Western blot技术检测lncRNA-CEPT8编码的小肽。结果表明:lncRNA-CEPT8全长为1 248 bp,在鸡原始生殖细胞质中富集,具有大小为246 bp且编码81个氨基酸的ORF, Western blot检测结果表明lncRNA-CEPT8编码一条大小约为9 ku的小肽。这一研究为进一步探究lncRNA-CEPT8编码的小肽在鸡原始生殖细胞中的调控作用及其对生殖细胞的影响奠定了基础。
In order to explore the regulatory role of lncRNA in chicken reproductive cells, we selected the fertilized eggs of Rugao yellow chicken as materials. Based on the results of high-throughput sequencing and screening in our laboratory, the full length of lncRNA-CEPT8 sequence was obtained by RACE technology. The enrichment location of lncRNA-CEPT8 was determined by quantitative fluorescence detection and nuclear plasmid separation experiments. ORF open reading frame prediction and construction of prokaryotic fusion expression vector were carried out. Western blot technique was used to find the evidence of the small peptide encoded by the lncRNA-CEPT8, which could provide a basis for further research on the role of the small peptide encoded by the lncRNA-CEPT8 in primordial germ cells. The results showed that the full length of lncRNA-CEPT8 was 1 248 bp, which was enriched in chicken primordial germ cytoplasm and encoded a small peptide about 9 ku in size. This study laid a foundation for further study on the regulation of protein in chicken primordial germ cells and the effect of lncRNA-CEPT8 on germ cells.
In order to explore the regulatory role of lncRNA in chicken reproductive cells, we selected the fertilized eggs of Rugao yellow chicken as materials. Based on the results of high-throughput sequencing and screening in our laboratory, the full length of lncRNA-CEPT8 sequence was obtained by RACE technology. The enrichment location of lncRNA-CEPT8 was determined by quantitative fluorescence detection and nuclear plasmid separation experiments. ORF open reading frame prediction and construction of prokaryotic fusion expression vector were carried out. Western blot technique was used to find the evidence of the small peptide encoded by the lncRNA-CEPT8, which could provide a basis for further research on the role of the small peptide encoded by the lncRNA-CEPT8 in primordial germ cells. The results showed that the full length of lncRNA-CEPT8 was 1 248 bp, which was enriched in chicken primordial germ cytoplasm and encoded a small peptide about 9 ku in size. This study laid a foundation for further study on the regulation of protein in chicken primordial germ cells and the effect of lncRNA-CEPT8 on germ cells.
引文
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[2] LI D,JI Y,WANG F,et al.Regulation of crucial lncRNAs in differentiation of chicken embryonic stem cells to spermatogonia stem cells [J].Animal Genetics,2017,48(2):191-204.
[3] DINAR Y,LETICIA A,FERREIRA D S L,et al.HIPSTR and thousands of lncRNAs are heterogeneously expressed in human embryos,primordial germ cells and stable cell lines [J].Scientific Reports,2016,6:327-353.
[4] HAMBURGER V,HAMILTON H L.A series of normal stages in the development of the chick embryo [J].Developmental Dynamics,2010,195(4):231-272.
[5] BAO J,WU J,SCHUSTER A S,et al.Expression profiling reveals developmentally regulated lncRNA repertoire in the mouse male germline [J].Biology of Reproduction,2013,89(5):107.
[6] LIANG M,LI W Q,TIAN H,et al.Sequential expression of long noncoding RNA as mRNA gene expression in specific stages of mouse spermatogenesis [J].Sci Rep,2014,4:59-66.
[7] 李碧春,陈国宏,赵东伟,等.鸡胚PGCs迁移与性腺发育关系的研究 [J].扬州大学学报(农业与生命科学版),2002,23(1):18-22.
[8] 金晓琳,胡福泉.利用3′-RACE法扩增到钙通道基因的3′-端片段 [J].细胞与分子免疫学杂志,1999(3):210-212.
[9] WANG M,ZHANG C,HUANG C L,et al.Regulation of fibroblast growth factor 8 (FGF8) in chicken embryonic stem cells differentiation into spermatogonial stem cells [J].Journal of Cellular Biochemistry,2018,119(2):- .
[10] DING J,YEH C R,SUN Y,et al.Estrogen receptor β promotes renal cell carcinoma progression via regulating IncRNA HOTAIR-miR-138/200c/204/217 associated CeRNA network [J].Oncogene,2018,37:5037-5053.
[11] LUK A C,CHAN W Y,RENNERT O M,et al.Long noncoding RNAs in spermatogenesis:insights from recent high-throughput transcriptome studies [J].Reproduction,2014,147(5):R131.
[12] 陈兴勇,杜叶叶,梁维炜,等.产蛋相关激素对脂质合成基因及卵黄受体基因的表达调控研究 [J].扬州大学学报(农业与生命科学版),2019,40(1):62-66.
[13] ANDERSON D M,ANDERSON K M,CHANG C L,et al.A micropeptide encoded by a putative long noncoding RNA regulates muscle performance [J].Cell,2015,160(4):595-606.
[14] ANGRAND P O,VENNIN C,LE X F,et al.The role of long non-coding RNAs in genome formatting and expression [J].Frontiers in Genetics,2015,6:165.
[15] XUE X,YANG Y A,ZHANG A,et al.lncRNA HOTAIR enhances ER signaling and confers tamoxifen resistance in breast cancer [J].Oncogene,2016,35(21):2746-2755.