绵羊CYP19基因卵巢启动子的克隆及其表达活性研究

doi:10.11843/j.issn.0366-6964.2015.09.008

Abstract

Abstract:

The research was conducted to clone the sheep CYP19 gene ovarian promoter fragments and detect the tissue-specific expression in cells level.According to the known sequence the specific prmiers were designed，1.1 and 0.5 kb of sheep CYP19 gene ovarian promoter fragment were amplified by PCR，then the sequences were analyzed by software with published sequences.After eukaryotic expression vector pCYP19-1.1-EGFP-N₂ and pCYP19-0.5-EGFP-N₂were constructed by cloning promoter fragments into the pEGFP-N₂ vector without CMV，then the recombinant plasmids were transfected into sheep granular cells and fetal fibroblast cells by liposome Lipofectamine ^TM LTX+PLUS.The EGFP fluorescence expression was observed under the microscope after transfection for 24，48 and 72 h.The sequenced results showed that sheep CYP19 gene promoter fragments which were cloned were 1.1 and 0.5 kb length and had highly homologous with the published sequences.The sequence analyzing with transcription factor binding sites prediction software indicated that the promoter fragment contained a core promoter cis-element smilar with TATA box and transcription factor binding sites.24 h after transferring with pCYP19-1.1-EGFP-N₂，the EGFP-expressing positive granulosa cells could be observed，48 h after transferring，the EGFP-expressing positive granulosa was increased，72 h after transfection the EGFP-expressing positive granulosa increased to the most.But after transfection the EGFP which was expressed in sheep fetal fibroblast cells was little.The results also showed that no EGFP-expressing positive granulosa cells and fetal fibroblast cells were observed at 24，48 and 72 h after transfection with pCYP19-0.5-EGFP-N₂.The sheep CYP19 promoter 1.1 kb can drive foreign gene expressing in sheep granulosa cells and it can be used for the functional studies of fecundity-related genes and transgenic animal research，but it is not ovarian specific expression promoter.

CLC Number:

S826.2

SUN Hong-xin，ZHANG Ying-jie，LIU Yue-qin，CHEN Xiao-yong,DUN Wei-tao. Cloning and Expression Assay of Sheep CYPl9 Gene Ovarian Promoter[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(9): 1540-1548.

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Cloning and Expression Assay of Sheep CYPl9 Gene Ovarian Promoter

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