华北农学报
華北農學報
화북농학보
ACTA AGRICULTURAE BOREALI-SINICA
2015年
2期
6-11
,共6页
猪%SIM1基因%启动子活性%表达模式
豬%SIM1基因%啟動子活性%錶達模式
저%SIM1기인%계동자활성%표체모식
Pig%SIM1 gene%Promoter activity%Expression pattern
旨在对猪SIM1基因的转录调控机制及表达模式进行初步探讨。采用PCR法扩增猪SIM1基因5′端上游的启动子区，应用生物信息学方法对这一区域内潜在的转录因子结合位点进行预测，采用5′端侧翼序列缺失获得8段长度不等的启动子片段，并分别克隆至荧光素酶报告基因表达质粒（ pGL3-Enhancer）中，利用双荧光素酶报告活性检测分析 SIM1基因启动子区不同长度片段在293T、MGC803和 Bel7402细胞中瞬时转染后的活性。同时，采用 Western Blot实验检测 SIM1蛋白在猪7个不同组织的表达模式。结果表明，各 SIM1启动子片段在293T、MGC803和 Bel7402细胞中均有活性，在293T 细胞活性最低；-699~-489 bp 存在关键顺式调控元件，这一区域发现 Smad、CEBPα、PAX6等关键转录因子结合位点；Western Blot实验首次在中枢神经系统外发现 SIM1的表达，SIM1蛋白在脑和下丘脑表达量最高，在睾丸的表达量次之，在肝脏、皮脂和甲状腺表达量较高，在肌肉表达量最低。研究结果提示，SIM1基因在神经细胞发育、脂肪代谢和性腺发育过程都可能起到重要作用。
旨在對豬SIM1基因的轉錄調控機製及錶達模式進行初步探討。採用PCR法擴增豬SIM1基因5′耑上遊的啟動子區，應用生物信息學方法對這一區域內潛在的轉錄因子結閤位點進行預測，採用5′耑側翼序列缺失穫得8段長度不等的啟動子片段，併分彆剋隆至熒光素酶報告基因錶達質粒（ pGL3-Enhancer）中，利用雙熒光素酶報告活性檢測分析 SIM1基因啟動子區不同長度片段在293T、MGC803和 Bel7402細胞中瞬時轉染後的活性。同時，採用 Western Blot實驗檢測 SIM1蛋白在豬7箇不同組織的錶達模式。結果錶明，各 SIM1啟動子片段在293T、MGC803和 Bel7402細胞中均有活性，在293T 細胞活性最低；-699~-489 bp 存在關鍵順式調控元件，這一區域髮現 Smad、CEBPα、PAX6等關鍵轉錄因子結閤位點；Western Blot實驗首次在中樞神經繫統外髮現 SIM1的錶達，SIM1蛋白在腦和下丘腦錶達量最高，在睪汍的錶達量次之，在肝髒、皮脂和甲狀腺錶達量較高，在肌肉錶達量最低。研究結果提示，SIM1基因在神經細胞髮育、脂肪代謝和性腺髮育過程都可能起到重要作用。
지재대저SIM1기인적전록조공궤제급표체모식진행초보탐토。채용PCR법확증저SIM1기인5′단상유적계동자구，응용생물신식학방법대저일구역내잠재적전록인자결합위점진행예측，채용5′단측익서렬결실획득8단장도불등적계동자편단，병분별극륭지형광소매보고기인표체질립（ pGL3-Enhancer）중，이용쌍형광소매보고활성검측분석 SIM1기인계동자구불동장도편단재293T、MGC803화 Bel7402세포중순시전염후적활성。동시，채용 Western Blot실험검측 SIM1단백재저7개불동조직적표체모식。결과표명，각 SIM1계동자편단재293T、MGC803화 Bel7402세포중균유활성，재293T 세포활성최저；-699~-489 bp 존재관건순식조공원건，저일구역발현 Smad、CEBPα、PAX6등관건전록인자결합위점；Western Blot실험수차재중추신경계통외발현 SIM1적표체，SIM1단백재뇌화하구뇌표체량최고，재고환적표체량차지，재간장、피지화갑상선표체량교고，재기육표체량최저。연구결과제시，SIM1기인재신경세포발육、지방대사화성선발육과정도가능기도중요작용。
To investigate the transcriptional regulation mechanism and expression pattern of porcine Single-minded 1(SIM1)gene. 5′ promoter fragment was amplified with PCR method and the potential transcription factor binding sites were predicted with bioinformatics method;Eight promoter segments with different length were obtained by promoter deletion analysis and cloned into pGL3-Enhancer vector,then the promoter activities were determined in transiently transfected 293T,MGC803 and Bel7402 cells by the dual-luciferase assay system,respectively. Mean-while,SIM1 protein expression pattern was detected in seven tissues using Western Blot( WB)method. The results showed that all these SIM1 promoters presented activities in 293T,MGC803 and Bel7402 cells,and the lowest in 293T cells. The region of -699— -489 bp contained the key cis-regulatory elements and further bioinformatics a-nalysis found some key transcription factor binding sits,such as Smad,CEBPα and PAX6. WB experiments revealed that,besides its known expression in central nervous system,SIM1 protein could be detected in all examined tis-sues. Grayscale analysis showed a strong SIM1 protein expression in brain,hypothalamus and testes,a weak to mod-erate expression in muscle,thyroid gland,subcutaneous fat and liver. These results indicated that SIM1 gene may play important role in nerve cell development,fatness metabolism and gonads development.
