Clone And Function Analysis Of Mads-Box Gene Involved In Ovule Development Of Seedless Grapevine

In this study, we firstly isolated the MADS-Box genes involved in ovule developement from a full length cDNA libraty of seedless grapevines abortive ovules. Then we studied their expression patterns in defferents organs and different ovule develoment stages using RT-PCR and Real-time PCR, respectively. On the basis, three differentially expressed genes involed in ovule and an ovule-specific expression gene were obtained. In order to analyzed and identification the function of these differentially expressed genes, the TRV-vector of the three genes were constructed and transformed into tomato. Accoding detected the infiltrated tomato mediated by Agrobacterium, we could indicated preliminarily the MADS-Box genes played an essential role in the regulation and control of seed development. Cloned the promoter sequence of the ovule-specific gene, and constructed its deletion vectors. The main results were as follows:1. Seven MADS-Box cDNA sequences were cloned from the ovule-abort cDNA library. They were named VtMADS1、VtMADS2、VtMADS3、VtMADS4、VtMADS5、VtMADS6、 VtSVP. Sequence analysis showed that, all the MADS-box genes contained MADS domain and K domain, belonged to the MICK-type MADS-Box gene. The phylogenetic analysis showed that these genes belonged to different MADS-Box subfamilies. Concrete was:VtSVP belonged to B class. VtMADS1, VtMADS2and VtMADS3belonged to C/D class. VtMADS4, VtMADS5and VtMADS6belonged to E class.2. These genes expressed differently in the grape tissue including root, sterm, leaf, bud, full bloom and ovule examined by RT-PCR. Among them VtMADS1gene only expressed in the ovule. VtMADS2, VtMADS3, VtMADS4, VtMADS5and VtMADS6genes expressed in flowers and ovule, but there were no expression in root, stem and leave. VtSVP gene was expressed in all of the tissues. Therefore, we got an ovule-specific expressed gene.3. Real-time PCR analysis revealed that the gene expression level during ovule development in Thompson and Pinot was different. The results indicated that the expression level of VtMADS1, VMADS2, VMADS4and VtMADS5genes were basically same, however, VtMADS3, VtMADS6and VtSVP gene were completely different. Therefore, we got three differentially expressed genes involed in ovule development.4. Make differentially expressed genes to builded the virus-induced gene silencing vector. To examined whether TRV-vector could be used efficiently to silence genes in tomato, we detected the target gene by RT-PCR. Compared with the control, the number of the tomato seed infiltrated with TRV-LeMADS was significantly decreased. The results showed that the LeMADS gene may be associated with fuirt seedlessness of tomato.5. PCR method was used to isolated the ovule-specific promoter. This promoter is1780bp. Promoter function prediction and cis-element analysis indicated that the sequences have many cis-elements connected with ovule developemnt such as:P-box, TATC-box, GCN4, Skn-1, GARE-motif The deletion vectors fused with GUS gene was transformed into Arabidopsis thaliana medicated by Agrobacterium, the seed was obtained which was helpful for further analysis of the promoter.