MRNA In Situ Hybridization With Genes Related With Lily Flowers Development

The mRNA In Situ Hybridization method is a very important molecular biological method for gene expression research on plants. In this study, based on the results about genes related with flowers development already, we researched the details in the process of In Situ Hybridization, and set up an experiment system which suitable for flowers of Lilium longiflorum. We used the genes LLGLO1 and LLTCP1 to synthesis RNA probes and hybridization with flowers of Lily to detect genes expression. The particular contents were follows:1. We compared the RNA in different samples which were treated with different methods. The result indicated that, RNA of 2 hours sample began to degradation. Therefore, we should treat the samples whthin 1.5 hours after they leave the maternal plants. Second, we extracted total RNA form samples which were treated by FAA, kano and 4% PFA. The result indicated that kano can conserve integral RNA, So when we treat samples, we can do the same as kano, ensure solutions are RNase free, and avoid RNA degradation by anthropogenic handling.2. We made the RNA probes. We inserted specific DNA subsequence into T easy vector, and synthesis RNA probes by in vitro transcription. After blot detection, the RNA probes are of high quality.3. We researched the adequate digest condition by proteinase K. The result indicated that, we can choose proteinase K of 10μg/mL to incubate for 20-30 minutes at 37℃.4. We compared the result of RNA In Situ Hybridization between paraffin section and frozen section. The result indicated that, tissues in paraffin section were complete, and the signals in tissues were apparent. However, tissues in frozen section were incomplete, and the cells'breaking may lead some signals lost. Also, we compared the result of RNA In Situ Hybridization between kano treated samples and FAA treated samples with paraffin section. The result indicated that, signal located in the same position of both methods treated samples, but the signal intensity were different. Kano treated samples may lose some signal during hybridization process.5. We used the antisense RNA probe to detect the expression of LLGLO1 and LLTCP1. The result indicated that, LLGLO1 were expressed in four whorls of floral-organ in Lilium longiflorum, and with the maturate of floral-organ, the express level were more; The expression of LLTCP1 were more likely similar with other TCP genes, the signal appeared in the parts of cell cleavage.