水牛YY1基因克隆及其shRNA片段的筛选

doi:10.11843/j.issn.0366-6964.2014.01.009

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (1): 62-68.doi: 10.11843/j.issn.0366-6964.2014.01.009

水牛YY1基因克隆及其shRNA片段的筛选

龚云，乔树叶，林浪，王梦，石德顺，李湘萍^*

(亚热带农业生物资源保护与利用国家重点实验室，广西大学，南宁 530004)

收稿日期:2013-07-18 出版日期:2014-01-23 发布日期:2014-01-23
通讯作者: 李湘萍，研究员，E-mail：xiangpingli@163.com
作者简介:龚云(1988-)，女，湖南长沙人，硕士，主要从事细胞与胚胎工程的研究，E-mail：283148675@qq.com
基金资助:
科技部“863”计划项目（2011AA100607）；广西自然科学基金回国基金重点项目（2012GXNSFCB053002）；国家留学回国基金项目（BLH120499）

Cloning of Buffalo YY1 Gene and Screening Its shRNA Fragments

GONG Yun，QIAO Shu-ye，LIN Lang，WANG Meng，SHI De-shun，LI Xiang-ping^*

（State Key Laboratory of Subtropical Bioresource Conservation and Utilization，Guangxi University，Nanning 530004，China）

Received:2013-07-18 Online:2014-01-23 Published:2014-01-23

摘要/Abstract

摘要：

本试验旨在克隆水牛阴阳因子1（YY1）基因，并筛选获得可有效抑制水牛YY1基因表达的shRNA干扰片段。以水牛成纤维细胞cDNA为模板，采用RT-PCR方法，扩增水牛YY1基因CDS序列，将其连接插入到pMD18-T载体中，进行序列测序并分析。采用Xho I和EcoR I双酶切，将水牛YY1基因编码区定向克隆至pEGFP-N1载体中，构建其真核表达载体pYY1-EGFP-N1。设计2条靶向水牛YY1基因的shRNA序列并构建其慢病毒表达载体，命名为pSicoR-GFP-YY1 shRNA1/2。以pSicoR-GFP和pSicoR-GFP-1864分别为空白对照和阴性对照质粒，采用脂质体转染方法，将YY1真核表达载体分别与2条shRNA慢病毒表达载体共转染293T细胞，48 h后观察绿色荧光蛋白EGFP表达情况；72 h收集共转染细胞样品，采用qRT-PCR和Western-blot方法分析YY1基因的表达水平。结果表明，克隆得到1 248 bp的水牛YY1基因CDS序列，其与牛YY1基因的同源性达99%。构建的水牛YY1基因真核表达载体pYY1-EGFP-N1能在水牛胎儿成纤维细胞（BFF）中瞬时表达。酶切分析及序列测定结果表明，所构建的shRNA慢病毒表达载体pSicoR-GFP-YY1 shRNA1/2为阳性克隆。质粒共转染293T细胞48 h后，可观察到绿色荧光蛋白EGFP的表达。qRT-PCR结果显示，设计合成的2条shRNA对水牛YY1基因的表达均有抑制效果，其中YY1 shRNA2的抑制效果（93.64%）显著高于YY1 shRNA1（50.77%）(P<0.05)，Western-blot分析结果显示，2条shRNA对YY1蛋白表达均有抑制效果。以上结果说明克隆得到水牛YY1基因编码区序列，并获得2条有效抑制其表达的shRNA片段，为进一步阐明YY1基因在水牛早期胚胎发育过程中的作用奠定了基础。

Abstract:

The aim of present study was to clone the CDS sequences of buffalo YY1 gene，and screen its effective shRNA fragments.The cDNA of buffalo fetal fibroblast(BFF) was used as the PCR template，the CDS full-length sequence of buffalo YY1 gene was amplified by using touch down RT-PCR methods.The purified CDS fragment was inserted into pMD18-T vector，and the positive plasmid was identified and sequenced.Fusion expression vector of buffalo YY1 gene was constructed by inserting the YY1 CDS fragment into pEGFP-N1 vector，named as pYY1-EGFP N1.The pYY1-EGFP-N1 plasmid was transfected into BFF cells by Lipofectamine^® LTX reagent.Two shRNA fragments targeting buffalo YY1 gene were designed and synthesized，their lentiviral expression vectors were constructed，named as pSicoR-GFP-YY1 shRNA1/2 respectively.YY1 recombinant plasmid (pYY1-EGFP-N1) and shRNA lentiviral expression plasmid (pSicoR-GFP-YY1 shRNA1/2) were co-transfected into 293T cells by using Lipo-LTX reagent at the ratio of 1:1 or 1:6，pSicoR-GFP and pSicoR-GFP-1864 plasmid were used as blank and negative control respectively.At 72 h after transfection，the cells were harvested，and the total RNA was extracted.The expression of YY1 gene in each transfected cells was detected by qRT-PCR and Western-blot methods. The results showed that the CDS sequence of cloned buffalo YY1 was 1 248 bp，the nucleotide homology of YY1 gene between bovine and buffalo was 99%.The constructed pYY1-EGFP-N1 plasmid could transiently express in BFF cells.The results of qRT-PCR showed that the two designed shRNAs could inhibit the expression of buffalo YY1 mRNA，the shRNA2 fragment had significantly higher inhibition effect than shRNA1 (93.64% v 50.77%) (P<0.05).The results of Western-blot analysis confirmed that the two shRNAs had inhibition effect on gene expression of buffalo YY1.The above results indicated that the CDS sequence of buffalo YY1 was obtained，two effective shRNA fragments targeting buffalo YY1 gene were selected，which laid foundation for further study the role of YY1 gene in buffalo embryo development.

中图分类号:

823.8⁺3.2

龚云，乔树叶，林浪，王梦，石德顺，李湘萍. 水牛YY1基因克隆及其shRNA片段的筛选[J]. 畜牧兽医学报, 2014, 45(1): 62-68.

GONG Yun，QIAO Shu-ye，LIN Lang，WANG Meng，SHI De-shun，LI Xiang-ping. Cloning of Buffalo YY1 Gene and Screening Its shRNA Fragments[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(1): 62-68.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2014.01.009

https://www.xmsyxb.com/CN/Y2014/V45/I1/62

参考文献

［1］AFFAR E，GAY F，SHI Y J，et al.Essential dosage-dependent functions of the transcription factor Yin Yang 1 in late embryonic development and cell cycle progression ［J］.Mol Cell Biol，2006，26(9)：3565-3581.

［2］DONOHOE M E，ZHANG X，MCGINNIS L，et al.Targeted disruption of mouse Yin Yang 1 transcription factor results in peri-implantation lethality ［J］. Mol Cell Biol，1999，19(10)：7237-7244.

［3］ZHENG P，PATEL B，MCMENAMIN M，et al.Expression of genes encoding chromatin regulatory factors in developing rhesus monkey oocytes and preimplantation stage embryos：possible roles in genome activation ［J］.Biol Reprod，2004，70(5)：1419-1427.

［4］GALVIN K M，SHI Y.Multiple mechanisms of transcriptional repression by YY1 ［J］.Mol Cell Biol, 1997，17(7)：3723-3732.

［5］PALMER M B，MAJUMDER P，COOPER J C，et al.Yin Yang 1 regulates the expression of snail through a distal enhancer ［J］.Mol Cancer Res，2009，7(2)：221-229.

［6］TAKASAKI N，KUROKAWA D，NAKAYAMA R，et al.Acetylated YY1 regulates Otx2 expression in anterior neuroectoderm at two cis-sites 90 kb apart ［J］.EMBO J，2007，26(6)：1649-1659.

［7］LEE J S，GALVIN K M，SHI Y.Evidence for physical interaction between the zinc-finger transcription factors YY1 and Sp1 ［J］.Proc Natl Acad Sci U S A，1993，90(13)：6145-6149.

［8］DONG X P，PFISTER H.Overlapping YY1- and aberrant SP1-binding sites proximal to the early promoter of human papillomavirus type 16 ［J］.J Gen Virol，1999，80(Pt 8)：2097-2101.

［9］YAKOVLEVA T，KOLESNIKOVA L，VUKOJEVIC V，et al.YY1 binding to a subset of p53 DNA-target sites regulates p53-dependent transcription ［J］.Biochem Biophys Res Commun，2004，318(2)：615-624.

［10］REZAI-ZADEH N，ZHANG X，NAMOUR F，et al.Targeted recruitment of a histone H4-specific methyltransferase by the transcription factor YY1 ［J］.Genes Dev，2003，17(8)：1019-1029.

［11］GRIFFITH G J，TRASK M C，HILLER J，et al.Yin-Yang1 is required in the mammalian oocyte for follicle expansion ［J］.Biol Reprod，2011，84(4)：654-663.

［12］KIM J D，KANG K，KIM J.YY1’s role in DNA methylation of Peg3 and Xist ［J］.Nucleic Acids Res,2009,37(17):5656-5664.

［13］WU S，HU Y C，LIU H，et al.Loss of YY1 impacts the heterochromatic state and meiotic double-strand breaks during mouse spermatogenesis［J］.Mol Cell Biol，2009，29(23)：6245-6256.

[1]	郭振伟, 黄永军, 范威宏, 文冬梅, 沈朋雷, 潘尔科, 陆凤花, 石德顺. 褪黑素对水牛卵母细胞体外成熟的影响及其受体介导机制的探究[J]. 畜牧兽医学报, 2019, 50(2): 314-322.
[2]	刘晓华, 文冬梅, 余庆, 邓凯, 陆凤花, 石德顺. 组蛋白甲基化抑制剂UNC0638对水牛转基因细胞外源基因表达的影响[J]. 畜牧兽医学报, 2018, 49(10): 2163-2169.
[3]	沈朋雷, 秦希玲, 尹倩, 郭振伟, 邓凯, 杜姗姗, 尹娜, 阮子芸, 石德顺, 陆凤花. 水牛卵母细胞GV/MⅡ期对应卵丘细胞miRNA差异表达分析[J]. 畜牧兽医学报, 2017, 48(5): 836-845.
[4]	雷小灿，张海航，崔奎青，刘福航，刘庆友，石德顺. 水牛BMP1基因对颗粒细胞增殖与凋亡的功能研究[J]. 畜牧兽医学报, 2015, 46(10): 1766-1774.
[5]	黄愉淋，付强，黄德伦，潘宏，黄凤玲，梁贤威，张明. 利用TMT标记结合2D LC-MS/MS技术分析不同时期水牛睾丸曲精细管差异蛋白质组[J]. 畜牧兽医学报, 2014, 45(8): 1265-1273.
[6]	黄愉淋，付强，黄德伦，陈富美，黄凤玲，梁贤威，张明，卢克焕. 去除高丰度蛋白质后水牛卵泡液的双向电泳差异分析[J]. 畜牧兽医学报, 2014, 45(7): 1113-1119.

水牛YY1基因克隆及其shRNA片段的筛选

Cloning of Buffalo YY1 Gene and Screening Its shRNA Fragments

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 6

编辑推荐

Metrics

本文评价