通过差减文库筛选文昌鱼神经胚中特异表达的基因

Screening of specifically expressed genes in amphioxus neurula by constrution of a subtractive cDNA library

目的:构建文昌鱼12 h神经胚与6 h原肠胚的差减cDNA文库,以期从中挑选出在神经、肌肉、体轴发育过程中特异表达的基因,以便对文昌鱼早期胚胎的发育调控机制进行进一步研究。方法:分别从文昌鱼12 h胚胎和6 h胚胎提取总RNA,反转录成cDNA,以文昌鱼12 h胚胎的cDNA作为tester,并以6 h胚胎的cDNA作为driver,经过两轮杂交和抑制性PCR扩增,产物与T载体连接,经蓝白斑筛选,提取质粒DNA,EcoR I酶切鉴定插入片段,由此构建文昌鱼早期两个不同发育时期的差减cDNA文库。结果:对200个文库克隆进行了测序,筛选到一些可能在文昌鱼12 h神经胚中特异表达的基因。结论:成功构建了文昌鱼12 h/6 h胚胎差减cDNA文库,所获得的12 h神经胚中特异表达的基因,为进一步对文昌鱼早期发育调控机制进行研究提供了材料。

AIM： To screen specifically expressed genes in the development of nerve,muscle,and body axis of amphioxus,Branchiostoma belcheri tsingtauenese.METHODS： A subtractive cDNA library was constructed from the 12-hour amphioxus neurula cDNA after subtractively hybridized with the 6-hour amphioxus gastrula cDNA.The total RNA was extracted from the 12-hour neurula and 6-hour gastrula,then reverse transcribed into cDNA.The 12-hour neurula cDNA was designated as the experimental group（the tester） and the 6-hour gastrula cDNA as the control group（the driver）.The differentially expressed sequences were exponentially amplified using suppression PCR.Background was subtracted and differentially expressed sequences were further enriched.The PCR products were ligated to the T Vector.After transformation of the recombinant plasmid carrying inserted amphioxus cDNA into E.coli host cells,the cDNA library was constructed successfully.RESULTS： Two hundred randomly chosen positive clones were sequenced and some of neurula-specifically expressed genes were obtained.CONCLUSION： SSH is an effective method for searching differentially expressed genes.The subtractive cDNA library we generated provides a tool for further study of regulatory mechanisms of amphioxus early embryonic development.