| In this study,based on the formal results about wood formation molecular biology,and the mRNA in situ hybridization with expansin gene of ClEXP2 anti-sense probe performed to detect gene expression during Chinese fir stem development.The main results were follows:1.The soft stem of Chinese fir genotype 020 collected for the paraffin sections skills optimization.Some modification were made,these made the tissue slice clear and integrity.2.It shown that the Endogenous Alkaline PhosPhatase(AP) in Paraffin tissue sections in our tests has rich blue-violet stain dots,which mainly distributed in pith,phloem and cortex,through NBT/NCIP colour development testing.It displays that Endogenous Alkaline PhosPhatase(AP) in Paraffin tissue sections is activity,which will affect next RISH,so it is necessary to strengthen blockade for Endogenous Alkaline PhosPhatase(AP).3.By the design of the specific primers with the 5',restriction site,the 280bp specific sequence from CLEXP2 gene amplified,in which T vector constructed with T7 promoter.After the plasmid positive clones treated through linearization, it used for a template to synthesis mRNA antisense probe of CLEXP2 gene with a marker of digoxigenin.??It was that,both the experimental groups and contrast groups had the similar purple-red spots,but for the experimental group,the samples have the hybridization signal only came from the antisense probe.The spots of the probe in blue-violet spots spread on the transverse sections across the pith,cambium, phloem and cortex.In conclusion,this thesis presented useful information for the expression patterns of expansin during the Chinese fir cambium cells processing in secondary growth,and establish the system of Chinese fir RISH successfully,which creates a platform for the research of differentially expressed genes. |
