Construction Of Subtractive Library Of Double-Single Flower Petunia And Functional Analysis Of Flower Development Genes

Petunia hybrida is an important material for gardening and study of plant development biology.. Intensive research has been done on the flower colour and flower pattern since the 1990s. Based on previous study, we carried out preliminary exploration on Petunia with regard to flowering time and formation of double-single flower. The final aim of our research was to explore the key genes involved in the formation of important traits, so that the genetic improvement of the species via genetic transformation could be implemented.The primary materials used in this study including double and single Petunia flowers obtained from segregated progenies through backcross were used for construction of SSH libraries; tobacco with double and single flower obtained from RNAi were used for another construction of SSH libraries; the in vitro tobacco plantlets was used for genetic transformation for characterize the function of new genes.The main results including:Firstly, using suppression subtractive hybridization (SSH) followed by differential screening through dot blot hybridization, totally 285 expressed sequence tags (ESTs) were identified. Analysis using Blastx searches showed that the libraries contained several important developmental genes. The expression patterns of selected genes, believed to be involved in the development of Petunia double flowers, were further analyzed by semi-quantitative or real-time reverse transcription-polymerase chain reaction (RT-PCR). These analyses were conducted using RNA extracts taken from specific floral organs and from flowers at various stages of development. Each of the selected genes showed differential expression levels between single and double flowers, particularly in young flower buds, suggesting that the genes are important for the early development of the double flower form. In particular, we discussed the expression patterns of genes with putative roles relating to cell expansion, organ fusion and organ number. Other genes, with less well-documented roles, may be involved in modulating the development of the Petunia double flower as well. Thus, such genes appear to function along with the major regulatory MADS-box genes as part of a complicated network of genetic factors that direct the formation of the Petunia double flower.In order to understand the expression pattern and functional mechanism of differentially expressed genes, we got the full length of PhFDH、PhHVA22、PhHSA, and constructed overexpression vector to investigate their functions. Based on our primary analysis, PhFDH might play some important roles in the formation of carpel-stamen fusion.Secondly, we also constructed single and double flower tobacco SSH libraries. A total of 251 expressed sequence tags (ESTs) were identified through dot blot hybridization. Analysis using homology search in MIPS Blastx database showed that the libraries contained several important developmental genes. The expression patterns of selected genes, indicated to be involved in the establishment of tobacco double flowers, were further analyzed by semi-quantitative and real-time reverse transcription-polymerase chain reaction (RT-PCR) in the different floral organs. All of the examined genes showed differential expression levels between single and double tobacco flowers. In particular we discussed the expression patterns of genes with putative roles relating to cell expansion, male sterility, carpel development. The data suggested that, in the NAGl disrupted double tobacco, some less well-characterized genes might be inactivated or activated by the disruption of NAG1 and then influenced the development of double tobacco flower.Thirdly, we the functions of FBP21 and FBP22, which belong to SOC1 like family in Petunia, were characterized. Both of the genes could promote flowering significantly. When FBP21 was ectopic expressed in tobacco, the main morphological characters were changed, i.e. the transformants was dwarfed, apical dominance was lost, lateral branches were more than wild type. Different from FBP21, transgenic FBP22 plants had no changes in flower organs compared with FBP21. The major differences in amino acid might be the cause of differential functions between FBP21 and FBP22.Finally, we cloned the full length of FT like gene in Petunia through in silico. Based on primary analysis about its composition of nucleotide and amino acid, it was assumpted that the gene was not the same as FT or TFL like genes. The result of functional characterization confirmed our assumption. This gene should be a new member of PEBP family existed in Petunia.