Analysis Of Different Expression Of Cdpks And Its Relevent Genes In The Dedifferentiation Of Mature Wheat Embryo

As it is known that CDPKs have recently been recognized as the key members in pathway of Ca²⁺ signals transduction and play essential roles in many processes of plant growth and development. To study functions of CDPKs in the dedifferentiation of mature wheat embryo, mature wheat embryos separated from seeds were used as explants. RNAs were extracted from embryos treated by 2,4-D in 0, 2 ,6, 12, 24 and 72 h. The Affymetrix Microarray technique was used to detect different expression of genes at each time-point. The expression data of cdpks and its relevant genes were analyzed and some key genes in analysis were verified by means of real-time PCR in hoping to elucidate CDPKs's possible functions in the dedifferentiation.The results showed that there were 15000 genes signiciantly changed in expression signal at the Microarray including 61127 probe sets, representing 55085 genes. 15 cdpks were found to be changed in expression pattern during dedifferentiation of mature wheat embryo, in which 12 genes were up-regulated by 2-7.5 times, 2 genes were down-regulated by 3-6.1 times, and 1 gene was up- or down- regulated at the different stages of dedifferentiation. According to the known functions of cdpks,their changed trend and the coincidence of the trend and the dedifferentiation degree, it was predicted that CPK2A, CPK2B , CPK6 and Os12g0169800 might play roles in the initiation of dedifferentiation of mature wheat embryos and the formation of calli.To further study the relation of cdpks and its relevent genes in the dedifferentiation of mature wheat embryo, 45 CDPKs target genes or upstream genes were also analyzed from the microarray. The results showed that their expression trends at different time points were consistent with those of CDPKs genes. By comparing different expression trends of CDPKs genes (CA654806 and BQ280973),14-3-3 protein gene (AF548740.1) and PM H⁺-ATPase (AY543630.1,or ha1) in the dedifferentiation, a skeleton pathway in Ca²⁺ signals transduction was put forward. Their relation should be like this: 14-3-3 protein CDPKs gen(eCA654806 and BQ280973) PM H⁺-ATPase.Real time PCR technique was employed to further verified BQ280973 and its target gene (ha1). We found that expression of these two genes were dynamic during the dedifferentiation of mature wheat embryo, and their number of copy appeared a maximum amount in the earlier stage (6 h) of the dedifferentiation, implying both of BQ280973 and PM H⁺-ATPase might take part in the initiation of dedifferentiation induced by aunxin (2,4-D). And the data of real time PCR was roughly consistent with microarray, verifying the results of microarray.The present paper studied on the dynamics of cdpks and their relavant genes during the dedifferentiation of mature wheat embryo at transcriptional level by using high-through wheat genes microarray technique. The functions of CDPKs in dedifferentiation of mature wheat embryo were emploitated at the first time. It is apparently a new idea to uncover the mechanism of the dedifferentiation of mature wheat embryo.