| Doritaenopsis hybrid obtained from the hybridization between Phalaenopsis and Doritis is famous for its long florescence, elegant flower types and vivid colors, which attracts the great interests of the customers and makes it become one of the widely used commercial cultivars in the market. However, a period under relative low temperature of 18-25? is often needed to induce its flowering, which mainly increases the cost and limits its market sale. Therefore, the key point to overcome the sensitivity to temperature is to investigate the molecular mechanism of how temperature regulates the flowering time in Doritaenopsis. In the present study, we tried to clone the target genes of DhAP2-1, DhAP2-2 and DhAP2-3 by RACE, and the interaction of DhMIR172 and target genes was analyzed. DhHMIR172 interference vector was constructed by use of Gateway technique which was subsequently transformed into protocorm of Doritaenopsis hybrid, This work will provide theories to regulate the flowering time of D. hybrid by transgenic ways. The main conclusions were as follows.1. Construction of RNA interference vector of DhMIR172DhMIR172 sequence cloned by our group before was used to design Gateway primers. The PCR amplified products were introduced into a directional connection site. Then the entry vector was constructed by use of the pENTRTM Directional TOPO(?) Cloning Kits. In the next, the objective sequence was transferred to the expression vector pHELLAGATE 8. Finally the RNA interference vector pHELLAGATE 8-DhMIRl72 was successfully obtained.The protocorm of Doritaenopsis hybrid 0908 was used as explant and transformed with Agrobacterium tumefaciens strains of GV3101. In order to verify the expression efficiency of constructed RNA interference vector, we used the 300 mg·L-1 Amp to inhibit the growth of A.tumefaciens and the spectinomycin was also applied to screen the regenerated plantlets. After one week of renewal cultivation, the A. tumefaciens broke out and the protocorm started to be browning. After two weeks, all tested protocorm showed brown and died. Up to now, we didn't get the transgenic plants.2. Target genes cloning of DhAP2-1, DhAP2-2 and DhAP2-3We obtained three target genes of DhAP2-1, DhAP2-2 and DhAP2-3 from Doritaenopsis hybrid by BLAST analysis to database of Phalaenopsis equestris. Based on the middle segment of DhAP2-I cloned in our group previously, we further got its whole length by 5' and 3' race. There was 2220 bp in the DhAP2-1 cDNA, containing the ORF region of 1479 bp, which encoded 492 amino acids with two AP2 conserved domains and DhMIR172 binding sites. Subsequently, the cDNA of DhAP2-2 and DhAP2-3 were amplified by RT-PCR with the whole length of 1058 bp and 738 bp , respectively. There was an ORF region of 1038 bp in DhAP2-2, encoding 345 amino acids and containing two AP2 conserved domain. Moreover, the DhAP2-3 contained an ORF region of 738 bp, encoding 245 amino acids w:ith one AP2 conserved domains.3. Dual Luciferase assay on DhM1R172 and target genes.We aimed to detect the luciferase activity after transfected the overexpression vector of co-transgenic wild clone of gene AP2-WT and miR172 mimic, co-rotational mutant clone of gene AP2-MUTA and miR172 mimic entering 293T cells. We compared and analysed the difference among the groups of co-transgenic wild-type clone of gene AP2-WT overexpression and NC mimic ,co-rotational mutant clone AP2-MUTA and NC mimic. , Compared with the miR172 NC group,AP2-1-WT group declined remarkably in s the luciferase activity. , indicating that DhAP2-1 could be interaction with miR172 in mRNA level. |
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