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目的:构建神经管cDNA文库,寻找神经管发育相关基因。方法:提取E8.5 d金黄地鼠神经管总RNA;SMART技术构建神经管cDNA噬菌体表达文库;重组噬菌体PCR鉴定。将出现频率很高的重组噬菌斑,经质粒转化、酶切鉴定和DNA序列分析,证实为高迁移率族蛋白B1基因(HMGB1)。将HMGB1 cDNA片段回收、纯化,制备探针;Northern杂交检测不同发育阶段神经管中HMGB1 mRNA的表达变化。结果:构建的HMGB1cDNA片段含有完整的cDNA序列。Northern杂交显示:随胚胎发育,神经管HMGB1 mRNA表达量逐渐增加,E10 d增加最为明显,E12 d仍处于较高水平;而8.5 d高温致畸胚神经管,HMGB1 mRNA表达量较对照组明显减少。结论:HMGB1基因的表达与神经管发育及高温致神经管畸形的发生密切相关。
Objective: To construct a neural tube cDNA library and search for neural tube development related genes. Methods: Total RNA was extracted from neural tube of golden hamster E8.5 d. The neural tube cDNA phage expression library was constructed by SMART technique. The recombinant phage was identified by PCR. The high frequency of recombinant plaques will be confirmed by the high mobility group box B1 gene (HMGB1) after plasmid transformation, restriction enzyme digestion and DNA sequence analysis. The HMGB1 cDNA fragment was recovered and purified to prepare probes. Northern blotting was used to detect the expression of HMGB1 mRNA in neural tube at different developmental stages. Results: The constructed HMGB1 cDNA fragment contains the complete cDNA sequence. Northern blotting showed that the expression of HMGB1 mRNA gradually increased with the increase of E10 d and remained at a high level at E12 d with embryonic development. However, the expression of HMGB1 mRNA in the neural tube and the neural tube of 8.5-day pyrogenic embryos were significantly decreased . Conclusion: The expression of HMGB1 gene is closely related to the development of neural tube and the occurrence of neural tube defects caused by hyperthermia.