Cloning And Functional Analysis Of A Mads-box Gene NtPR In Tobacco(nicotiana Tabaccum)

MADS-box genes play very important roles in plant development. The first MADS-box genes were cloned from yeast, Arabidopsis thaliana, Antirrhinum majus and human named MCM1, AGAMOUS, DEFICIENS and SRF respectively and hence MADS-box gene. MADS-box genes of plants have been extensively studied.In this project we cloned a MADS-box gene named NtPR, which belongs to the SVP subfamily. Blast result shows that there is over 80% homology on the protein level between NtPR and JIONTLESS of tamato which controls the development of pedicel abscission zone. Our data reveal that NtPR can affect the development of pedicel, a novel fuction for MADS-box genes. On the basis of the whole coding sequence obtained by rapid amplification of cDNA ends (RACE) assay, we further studied the function of this gene.(1) Taking the Arabidopsis thaliana leaves as materials, we carried out the sublocalization assay. Our data show that NtPR only express in the nuclei.(2) Many papers reveals that MADS-box gene usually form polymers, so we study the functional model of NtPR. Our data shows that this gene may form homo-dimers.(3) In our study, we obtained the NtPR over-expression and RNAi plants, both of which were driven by 35S promoter. Phenotypic analysis showed that over-expression plants developed shortened pedicels and the overall inflorescence structure becomes compact with early flowering. In contrast, inflorescence structures of RNAi plants become extended and the number of flowers reduced.(4) In over-expression transgenic plants, we found that NtBP, NtER, NtYUC were changed at the transcriptional level using real-time PCR. NtYUC gene is known as a key factor to auxin synthesis process. We then tested the expression of the auxin marker gene NtParC. The expression of NtParC was significantly increased. So we suggest that NtPR may be involed in auxin metabolism or signaling. (5) On the basis of results obtained above, we developed NtBP overexpression, NtBP RNAi and NtER antisense plants. The results show that pedicel elongation was affected in NtBP RNAi plants and the inflorescence becomes compact, while in NtPR over-expression plants the NtBP expression is down regulated. So we predict that NtBP is a downstream factor of NtPR in pedicel development signal pathway, although this hypothesis needs to be further investigated.NtPR impacts the development of pedicel as a member of MADS-box gene family. The pedicel may be controled by several other genes which play parallel roles as NtPR, explaining why in our RNAi lines we could not observe obvious phenotype. Our data also show that the NtPR may be involed in the auxin biosynthesis pathway. In addition, NtBP may play as a downstream member of NtPR regulating in pedicel development.