摘要
【目的】转录调控对于明确昆虫许多生理过程如抗药性、抗病性、行为、进化及发育等方面的机理具有重要意义。家蚕PTTH是调控生长发育的重要神经肽,明确其转录调控机理对阐明其表达的发育和组织特异性具有重要价值。【方法】本文克隆并测定了家蚕PTTH启动子序列,利用Matinspector分析软件预测该片段可能包含的顺式作用元件,采用高糖抽提缓冲液抽提家蚕脑核蛋白;标记PTTH启动子片段进行凝胶阻滞分析。【结果】家蚕PTTH启动子分析含有MEF2、TATA box、pbx-1等元件;抽提获得理想的脑核蛋白,脑核蛋白可以与PTTH转录起始位点附件119bp的序列特异地结合,把TATA Box的TATATAA突变后,脑核蛋白与突变后的探针MBS2不能结合。【结论】表明本研究中抽提的脑核蛋白是有效的,首次检测家蚕PTTH启动子可以与转录因子特异结合,初步鉴定了TATA Box。
[Objective] Knowledge of gene transcriptional regulation is a critical component of understanding many issues relevant to entomology, including pesticide and disease resistance, behavior, evolution, and development. Prothoracicotropic hormone （PTTH） is one of the key player in regulation of insect development and growth. Properties of transcription regulation of PTTH are assumed .to be an increasingly important role in elucidating the molecular basis of developmental and tissue specific expression pattern in Bombyx mori. [Method] In this paper, the promoter of PTTH gene was cloned and sequenced, then, the transcription regulation elements in the promoter were predicted using Matinspector software. The nuclear proteins were extracted from the brains of sile wormusing high sucrose solution. Electrophoretic Mobility Shift Assay was used to analyze the binding affinity of nuclear extraction to [α-^32p]dATP labeled probe. [Result] Some deduced cis-acting DNA elements were located in the promoter of PTTH gene of B. mori, including MEF2, TATA box, pbx-1, etc. The nuclear proteins were extracted from the brains, which can bind to the 119 bp DNA fragment near the transcriptional initiator of PTTH gene. When the 7 bp （TATATAA） of TATA Box was mutated （named MBS2）, the probe can not bind to the nuclear proteins. [Conclusion] The nuclear proteins extracted were effective. It is demonstrated that the promoter can bind to the nuclear proteins of B. mori, firstly. The TATA Box in the promoter of PTTH gene was identified preliminarily.
出处
《中国农业科学》
CAS
CSCD
北大核心
2008年第1期265-271,共7页
Scientia Agricultura Sinica
基金
国家自然科学基金(30500374)
国家重点基础研究发展计划“973”项目(2005CB121000)
教育部新世纪优秀人才基金(2007)
关键词
核蛋白
凝胶阻抑分析
PTTH基因
家蚕
Nuclear proteins
Electrophoretic mobility shift assay
Prothoracicotropic hormone gene
Bombyx mori
