斑茅两个看家基因片段的克隆及其在基因芯片中的应用

Cloning of Two House-keeping Genes from Erianthus arundinaceus and the Application in cDNA Microarray

根据已发表的同源基因序列,利用RT-PCR技术分离了斑茅(Erianthus arundinaceus)的GAPDH和APRT两个看家基因片段,用它们作为cDNA芯片阳性参照,以未经聚乙二醇(PEG)胁迫处理的斑茅叶片为对照,和PEG胁迫的4组材料同cDNA芯片进行杂交分析。杂交结果显示,GAPDH杂交的Cy5与Cy3平均信噪比(Signal/Noise,S/N)分别为56.12和60.8,APRT杂交的Cy5与Cy3平均信噪比分别为51.06和47.25,信噪比均很高;同时两个看家基因的杂交都显示出极强的信号,其中GAPDH的杂交信号值大于10000,APRT也在8000以上,杂交结果可靠。分析了PEG胁迫4个时段BADH与两个看家基因的表达,BADH的表达有明显变化,而看家基因表达均较稳定。上述结果表明所克隆的两个看家基因在斑茅中表达量高,且PEG胁迫下表达较为稳定,是基因芯片理想的阳性参照。

Positive controls were important for quality identification in microarray analysis of gene expression profiles. Two most important house-keeping genes GAPDH and APRT in plant often serve as positive standards. Here partial cDNAs of GAPDH （GenBank accession number： EF189713） and APRT （GenBank accession number： EF189712） were amplified from Erianthus arundinaceus by reverse transcriptase-polymerase chain reaction （RT-PCR） using two pairs of primers designed according to the corresponding sequence ofZea mays. Four sets of E. arundinaceus under PEG-stress were applied to the cDNA microarray hybridization using the two genes as positive controls. Profiles showed that the average values of singal to noise （S： N） of Cy5 and Cy3 were 56.12 and 60.8 in GAPDH, respectively, while 51.06 and 47.25 in APRT, respectively. High signal was detected in all the hybridization, with the values of over 10 000 in GA PDH and 8 000 in A PR T, indicating reliable hybridization. Both GA PDH and A PR T expressed more stably than BA DH. It is suggested that the two house-keeping genes from E. arundinaceus are suitable for positive controls in genechip applification.