Construction Of Yeast One-Hybrid Library And Gene Expression Profile In Double Petunia And Tobacco

Petunia hybrida is one of the most popular model plants. Recently, scientists have done robust research on its transcription factors related to the flowering development. The majority of those transcription factors belong to MADS-box family. In Arabidopsis, their functions have already been unveiled thoroughly, especially AG gene, which exhibiting type-C function. Researchers found that the loss-of-function in AG gene could lead to the conversion of stamens to petals, and the interminate development of floral meristem, which eventually result in the phenotype of double-flowered.We also found that transgenic tobacco exhibited double flowered when PMADS3of petunia was introduced via RNAi pattern. To explore the pathway in which PMADS3controls the development of double flower, we used wide type petunia (including single flowered and double flowered) and tobacco, transgenic tobacco as material, via the technique of gene microarray, suppression subtractive hybridization and real-time PCR, to screen the homologous genes that expressed differently between single and double flowers. Those genes were confirmed to be regulatory factors involved in the pathway and have controlled the formation of single/double flower. Furthermore, in order to better investigate the details, the yeast one-hybrid library was constructed which was aimed to figure out the relationship between those regulatory factors and to explore the unknown regulatory factors. The main results are listed below:1) We have detected the relative expression quantities of transcription factors (18in tobacco and33in petunia), whose sequences were downloaded from the website of NCBI, in the single and double-flowered plants by the method of real-time PCR. We have acquired15transcription factors expressed differently in petunia and15in tobacco, respectively. After the phylogenetic analysis, four pairs of homologous genes FBP20and Nttobmadsl, PMADS3and NtMADS3, FBP6and NtFBP6, PMADS12and NhSEP, were identified. Three pairs of them (except PMADS3genes) are inferred as candidates for the regulatory factors involved in the pathway controlled by PMADS3.2) After the analysis of petunia and tobacco’s gene microarray, differentially expressed genes whose Iog2value>2were clustered. They were divided into two groups: up-regulated genes and down-regulated genes in double flower. For the result of suppression subtractive hybridization, we took those genes expressed uniquely in double plants as up-regulated genes and those genes expressed uniquely in single plants as down-regulated genes, then group those genes as well. Combined the results, it is found that many petunia’s differentially expressed genes were highly homologous to those in tobacco, and their changing tendencies are identical.3) Those homologous differentially expressed genes achieved by above techniques were further testified by real-time PCR. From the result, we found that three pairs of homologous genes were all down-regulated in the double flowered plants. Therefore, we believed that these genes might function as regulatory factor during the development of single/double-flowered.4) To explore the role of those transcription factors and differentially expressed genes and figure out their relationship, we used the flower buds of double flower petunia to construct a Yeast One-Hybrid AD-cDNA Library. This library has been evaluated via transformation efficiency and the length of recombination fragments.