Molecular Mechanism Of The Flower Bud Abortion And Anther Development In Petunia

Flower abortion is always one of the most important factors affecting production and ornamental traits of the horticultural crops.Researches on the molecular mechanism of flower abortion will help us to improve the crop production and ornamental values.The normal development of anther is critical for the plant male feitlity,and the anther specific expressed genes usually play important roles in this process.Thus the study of these genes expression and regulation mechanism is of great scientific importance in exploring the regulation network of anther growth.Along with the improvement of the biological technology and the development of systems biology,both differentially expressed genes identified by suppression subtractive hybridization（SSH）and anther development patterns analysed by multi-omics will contribute to a more comprehensive explaination of the related biological phenomenon.Known as the"king of parterre flowers"and a model plant used in flower development research,Petunia hybrida was chosen to be studied on flower abortion and anther development.The key results are as follows:1.Construction and screening of the SSH librariesThe SSH was performed to compare the gene expression profiles between normal and abortive petunia flower buds as induced by short light periods.Using reverse northern blotting,a total of 912 positive clones were successfully identified and sequenced,and289 non-redundant,differentially expressed transcripts were obtained.According to the Blast2GO and KEGG pathway analyses,the significant distinct cellular component was identified as mitochondrion,and the notable different metabolic pathways were ascribed to carbohydrate metabolism and lipid metabolism.The quality of the SSH libraries was verified through quantitative real-time reverse transcription-PCR（qRT-PCR）analysis of14 genes.In addition,six of these genes were demonstrated to be of temporal and/or spatial specificity using qRT-PCR,implying that they might be responsible for the premature flower abortion.The data presented here provide insight into the molecular mechanisms of premature flower abortion under short light periods and,hence,may be of value for assessing candidate genes with potential for creating novel germplasms with enhanced photoperiod stress tolerance.2.Functional characterization and transcriptional regulation analysis of the PhGRPIn the previous study,a novel gene encoding a glycine-rich protein in Petunia hybrida（PhGRP）which obtained from the SSH library was found to be exclusively expressed in anthers,and its expression peaked earlier than that of the well-known tapetum-specific gene TA29 in tabacco and several genes in petunia with the classic anther-box cis-regulatory element.Furthermore,male fertility was significantly reduced in PhGRP-overexpressing lines driven by Ca MV35S constitutional promoter and the completely male-sterilty petunia without influence on other organs development have also been created driven by its own promoter with the BARNASE gene.Two transcription factors（TFs）which controlling the anther specific gene expression have also been selected by the Yeast one-hybrid library screening and the Dual luciferase experiment.In this research,the function of PhGRP gene has been explored,the regulatory mechanism of early anther-specific expression has been preliminary unravelled,novel gene resources and molecular strategies have been available for creating new male sterile lines.Therefore,this research is of great scientific importance and huge practical potentials.3.Identification of the members of the Dof and CYP77 gene familiesThrough expression analysis of the 23 Dof gene family members in petunia,we found that most of them were highly expressed in the floral.Some of them were nearly similarly expressed in the four whorls of the flower,however,some of them were specially expressed in one of the four whorls and showed temporal specificity.Furthermore,we found many Dof motifs in the promoter region of the PhGRP and a Dof gene was also obtained in yeast one-hybrid library.According to these,we suggest that the Dof family could play important role in the floral development and it supplies the important roles in exploring the function of the Dof family and the regulation network of the flower development.As a Phcyp77B1 gene was obtained from the SSH library,in order to further understand the function of CYP genes in plant,two cyp77 genes（Phcyp77A1 and Phcyp77B1）with the typical characteristics of the CYP77 family,were isolated from the petunia（Petunia hybrida‘Fantasy Red’）.Real-time quantitative polymerase chain reaction showed that they were both highly expressed in the flower organs,especially in the petals,while with lower levels in vegetative tissues and other three floral whorls.Both of their expression levels were related with the bud and petal development,indicating that they could play different roles in the floral development processes of petunia.Now,phenotype of the T₁ Phcyp77B1-RNAi plants is under identification.4.Transcriptomic and metabolomic analysis of the anther developmentThe anther development processes were analysed by a combined way of transcriptomics and metabolomics.Results showed that the glucose and fructose were most significantly changed metabolites,the photosynthesis pathway was one of the most important metabolic pathways.The anthers could control the photosynthesis speed by regulating the relative genes expression.In addition,51 kinds of transcription factors were found among the significantly changed genes,idicating that the anther development process is very activity,which deepened our understanding of the mechanism in petunia anther development.