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

Abstract

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.

CLC Number:

823.8⁺3.2

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.

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Cloning of Buffalo YY1 Gene and Screening Its shRNA Fragments

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