Functional Analysis Of Phased Development Related Gene FLC In

Citrus is an important economic crop. The long juvenile period, usally6-8years in citrus, has severely obstacled the breeding progress and genetic study on economically important traits, such as fruit quality and maturity. Previous studies indicated that trifoliate orange has to experience low temperature prior to flower formation, thus FLC gene might be involved due to its well known key important role in vernalization pathway. A FLC homolog, named PtFLC has been cloned from Poncirus trifoliata in our laboratory. Further experiment revealed that PtFLC showed alternative splicing into five transcripts selectively expressed in distinct developmental stages. In order to uncover the function of PtFLC, we constructed the RNAi vector, and then transformed it into wild trifoliate orange to create a material of down-regulated expression of PtFLC. The main results are as follows:1. RNAi vector Construction and transformation of Poncirus trifoliataSequence alignment indicated that the first exon in5’end of PtFLC was conserved among five transcripts. Thus, a209bp fragment of this part was chosen for target interference. Recombination sites were added to the ends of the. target fragment, and then the fragment was cloned into the interference vector pHGRV using Gateway technology. This vector can coded ihpRNA structure, and have the higher interference efficiency.The interference vector was transformed into wild trifoliate orange by agrobacterium-mediated transformation, and then through regeneration and resistance screening culture, ultimately six positive lines were obtained.2. PtFLC expression analysis in inteference plantsReal-time quantitative technology was used to analyze the expression of PtFLC in six transgenic lines, and the wild-type trifoliate orange as control, Reduced PtFLC transcription was observed in transgenic lines LI9, L84, L85, and L88.The L84and L90was the lowest, only0.2times of the control. This result confirmed that the target gene was silenced successfully3. PtSOCl and PtFT expression analysis in inteference plantsAt present, transgenic seedling is still at the juvenile stage. Phenotype observation showed that there is no difference between the inteference plants and wildtype.The expression level of PtSOC1and PtFT were investigated through Real-time PCR. The results showed that PtSOC1and PtFT expression highly increased in transgenic plants, showing the significantly negative correlation with PtFLC expression. This result confirmed that PtFLC influenced the downstream gene expression, and PtFLC was successfully silenced in trifoliate orange.the more phenotype of transgenic need to observation.