Functional Analysis Of Poncirus Trifoliata (L.) Raf PtFRI Gene Influencing Plant Flowering

The longer juvenile period of citrus is a problem that hinders citrus breeding.End of the juvenile period suggests that the plant’s transition from vegetative growth to reproductive growth(transitions of citrus flowering)and plant can flower normally.Therefore,the study of juvenile period has important biological significance.In the process of transforming from vegetative growth to reproductive growth,plants need to experience a series of changes in the external environment(temperature,light hormones,etc.),and there are complex gene regulatory networks.Studies have reported that FRI genes in Arabidopsis model plants mainly affect their flowering time through the low temperature vernalization pathway.Previous research in this laboratory found that citrus’ s flower bud differentiation needs to go through the low temperature.so this study is about the role of PtFRI gene in the low temperature vernalization pathway,the main results are as follows:1.Based on the citrus genome-wide genetic database and the FRI-like genes were compared and phylogenetically analyzed.It was found that PtFRI gene has the highest affinity with Arabidopsis thaliana FRI gene.The homology of the amino acid sequence is 85%.The cDNA of the FRI gene was cloned from the Trifoliate orange.The full-length cDNA was 1857 bp and encoded a total of 618 amino acids.2.The FRI gene was cloned from the Trifoliate orange and the pBI121 overexpression vector was constructed.The ectopic expression of PtFRI in Arabidopsis was late flowering compared to the wild type strain.qPCR analysis of the transgenic lines revealed that the expression of FRI and FLC was significantly up-regulated,and the expression of FT was significantly inhibited, indicating that FRI can inhibit the expression of FT and promote the expression of FLC and inhibit plant flowering.3.Subcellular localization of PtFRI gene fused to GFP revealed that PtFRI protein is mainly localized in the nucleus,whereas the control GFP protein is widely present in the nucleus and cytoplasm.This is similar to the previously reported Arabidopsis thaliana FRI gene mainly localized in the nucleus.4.qPCR analysis of different tissue of different citrus varieties showed that PtFRI gene expression was significantly higher in flowers and leaves.The GUS gene was ectopically expressed in Arabidopsis with the PtFRI promoter,GUS staining showed that the GUS gene was expressed with Arabidopsis throughout the developmental stages,but its expression gradually weakened with growth and development.At the stage it is mainly expressed in flowers and leaves.q PCR and GUS staining showed that the PtFRI gene is mainly expressed in the flowers and leaves of plants5.Vernalization of T3 transgenic seedlings revealed that the expression of GUS gene was significantly inhibited immediately after vernalization,and the GUS gene expression was significantly increased with the prolonged growth time under normal conditions.This shows that vernalization will inhibit the expression of PtFRI gene,but low temperature vernalization can not always inhibit the expression of PtFRI gene.In order to find transcription factors that bind to low-temperature elements,a bait vector was constructed as a template to perform a Yeast one-hybrid screening library.6.Four 5’deletion promoters vector were constructed and named as Q1-Q4.Q1-Q4 were transiently expressed in N.benthamiana and callus respectively.Only the Q4 promoter could initiate the expression of GUS gene,suggesting that the active site is located at transcription initiation site upstream of-771 bp.