罗氏沼虾18SrRNA基因生物素标记探针的制备及应用

探针(probe)是带有标记的特定DNA(RNA)片段,是核酸杂交鉴定特定基因以及研究特定基因组织表达的重要工具1]。PCR标记法是近年来发展起来的酶促标记基因探针的方法之一2]。常用的标记物有放射性物质(如γ 32P)和非放射性物质(地高辛,生物素,荧光素等)2]。在非放射性的标记物质中,生物素由于具有较高的化学稳定性和使用安全等优点而受到瞩目3]。在研究特定基因组织表达的过程中,由于仪器精确度限制和操作误差等因素,使用于杂交的各样品上样量难以达到绝对一致。如没有内参照杂交信号间的可比性就会降低而影响实验结果的可靠性。脊…

Preparation and application of the biotin-labeled probe of 18S rRNA gene in Macrobrachium rosenbergii

Probes are essential for study of gene expression and regulation. In this study, a method was established to prepare the biotin- labeled probe for 18S rRNA gene of freshwater prawn, Macrobrachium rosenbergii . And the labeled method was used to produce a lysozyme gene probe, then applied in analy sis of lysozyme gene expression. Primers were designed according to the nucleotide sequences of 18S rRNA of Decapoda in order to isolate the 18S rRNA gene sequences of M. rosenbergii. Total genomic DNA was isolated from hepatopancreas of the freshwater prawn. A specific DNA fragment with desired size was amplified by PCR using the total DNA as templates. The DNA fragment was inserted into pGEM- T Easy vector and sequenced. The result of BLAST and alignment analy sis confirmed that the DNA fragment iso lated was the 18S rRNA gene of M. r osenbergii, which was 418 nt in length. Biotin- labeled probe of the 18S rRNA was then produced by PCR using the recombinant plasmid as templates. The biotin-21- dTTP and the non- labeled dNTP were added to the PCR reaction system. Ratio of the biotin- 21-dTTP and the non- labeled dTTP was 3 to 1. The yield of the labeled probe is 300 ng#LL- 1. The detection limit of the probe is 60 pg. A biotin- labeled probe of ly so zyme gene was prepared by the same label method, and the yield of the lysozyme gene probe is 500 ng#LL- 1. These biotin- labeled probes were applied in Northern dot blotting analysis of tissue distribution of lysoyzme mRNA of M. rosenbergii. Signals were scanned and quantified by Analy sis System of Biolo gy Image. The signal intensity ratio of the lysozyme to 18S rRNA represents the relative expression level of lysozyme mRNA. The results showed that the lysozyme mRNA existed in all the tissues checked, including eye, muscle, gill, hepatopancreas, haemocytes and intestine. But lysoy zme mRNA levels varied among different tissues. The highest level was found in the intestine, and the second was in the hepatopancreas and the lowest was in the muscle. The signal intensities of 18S rRNA among tissues were consistent, which showed that the 18S rRNA gene expressed stably in different tissues and could be used as an internal standard for researches of specific gene expression in prawns.