Cloning And Functional Analysis Of SlAGO7in Tomato

ARGONAUTE (AGO) proteins were highly conserved and large gene family. Asthe core components of the silencing complex, they are integral players in all knownsmall RNA-directed regulatory pathways. Trans-acting siRNA (ta-siRNA) are a class ofendogenous siRNAs that can target mRNAs to control plant growth and development.AG07is key component of the TAS3ta-siRNA pathway. AGO7act function in plant byta-siRNA pathway. It was showed that the ta-siRNA pathway plays a conserved role inthe determination of lateral organ polarity. However, the contribution of the ta-siRNApathway in controlling tomato leaf and floral development is largely unknown.Therefore, Cloning and functional analysis of AGO7homologue SlAGO7in Solanumlycopersicum will further illuminate the role of ta-siRNA pathway in the regulation ofplant growth and development.To investigate the potential biological function of SlAGO7in plant development,the following aspects have been carried out in this study:1. Based on EST sequences from tomato genome database (SGN), the full-lengthcDNA and genomic DNA sequence of SlAGO7were isolated by using RACE and PCR.The bioinformatics methods were used to analyze the sequence characteristics ofSlAGO7. These results showed that the cloned full-length cDNA of SlAGO7was3238bp in length, containing a3003bp open reading frame (ORF) which encodes1000amino acids. In addition, the full length of genomic DNA contained three extrons andtwo introns. The deduced SlAGO7protein with two conserved PAZ and PIWI domainshad the typical characteristics of AGO family. This deduced amino acid had a highidentity with the known AGO7protein. Phylogenetic analysis showed that SlAGO7wasclassified into the ZIPPY/AGO7subfamily of AGO family.2. Real-time PCR method was used to investigate the temporal and spatialexpression pattern of SlAGO7gene. These results indicate that the expression of theSlAGO7gene was detected in vegetative tissues such as roots and stem, but not inleaves. SlAGO7mainly expressed in flower organ (Bud and flower), while its mRNAwas not detected in fruit. Furthermore, SlAGO7was found to be expressed in all theparts of flowers at anthesis stages. The expression level of SlAGO7increases slightly insepal and decreases dramatically in stamen. These data suggest that SlAGO7haspotential function to regulate plant growth and floral development in tomato. 3. To address the biology function of SlAGO7, the overexpression vectors andantisense vector were constructed. The plant expressive vector was transformed intotomato by Agrobacterium-mediated method. Overexpression of SlAGO7results insevere developmental defects in leaf and floral organs. Furthermore, SlAGO7overexpression altered transcript levels of TAS3ta-siRNA and its target genes. Thesedata indicate that SlAGO7regulate lateral organ morphology through the ta-siRNApathway.4. Overexpression of SlAGO7altered expression of a number of HD-ZIP III andMADS-box genes, indicating that they may directly or indirectly regulated by SlAGO7which contributed to control lateral organ morphology in tomato.