Characterization And Function Identification Of Citrus LIP2A In Tne Regulation Of Flowering

Citrus fruit trees have a juvenile period,it about 6-8 years.Overexpression of FLOWERING LOCUS T（FT）can greatly shorten the juvenile period of citrus.When the transgenic Arabidopsis thaliana which overexpressing FT gene is grafted as a rootstock,the FT protein move to the scion and cause early flowering of the scion.The early flowering of the scion is not observed when grafts on transgenic apples which overexpressing FT gene.Studies report that Jatropha which can induce early flowering of grafted scions has extremely high oil content.LIPOYLTRANSFERASE2（LIP2A）is a regulatory gene in the process of plant lipid metabolism.This gene is involved in the growth and development of plants,and also plays an important role in resisting environmental stress.Studies show that liposomes can regulate the movement of FT proteins and FT can specifically bind lipids.However,the function of the citrus LIP2A gene and its relationship with FT have not yet been elucidated.In this project,CcLIP2A is cloned from Clementine,we analyze its function.We explore the characterize of CcLIP2A in citrus flowering and its relationship with FT.The main work and test results are as follows:1.The CcLIP2A gene contains a lipid protein domain,phylogenetic tree analysis showed that LIP2A is highly conservatism in citrus.2.The 35S:CcLIP2A-EGFP and 35S:CcLIP2A-RFP-PtFT-EGFP overexpression vectors were transiently transformed into Nicotiana benthamiana.The results of sub-cellular localization showed that CcLIP2A protein is distributed in the endoplasmic reticulum.CcLIP2A and PtFT are co-expressed on the membrane.Using c DNA from different organs of citrus as template for q RT-PCR experiment,it find that the expression of CcLIP2A is higher in the stems and flowers.3.The overexpression of CcLIP2A in wild-type Arabidopsis showed an early flowering phenotype.The statistical results show that the number of rosette leaves in the T₂generation transgenic seedlings are significantly reduced and the flowering time is significantly earlier.The 35S:CcLIP2A-RFP-PtFT-EGFP vector is stably transformed into wild-type Arabidopsis to obtain transgenic lines.The result shows that there is no significant difference in flowering time between the T₁generation transgenic line and 35S:PtFT transgenic Arabidopsis.4.The wild lemons are cold treated at 4°C for 0 h,3 h,6 h,9 h,12 h,24 h,36 h,and 48 h.Quantitative result shows that the CcLIP2A responds to low temperature.At the same time,the VIGS lemon seedlings are freezing-treated at-4°C,and it find that the silent line is more sensitive to low temperature.5.The pGBKT7-CcLIP2A and pGADT7-CcLIP2A vectors are constructed,and the yeast two-hybrid experiment of CcLIP2A and PtFT is performed.The result shows that the four-deficient medium do not grow the yeast spots.We construct pET32a-CcLIP2A vector and do Pull-down experiment with PtFT,the result shows there is no interaction protein bands.Both of the two experiments show that PtFT and CcLIP2A do not interact in vitro.We construct 35S:CcLIP2A-FLAG and35S:PtFT-HA vectors for CO-IP experiments,the result shows that PtFT and CcLIP2A do not interact in vivo.In summary,The overexpression of the CcLIP2A in the wild-type Arabidopsis result in an early flowering phenotype.Therefore,the CcLIP2A gene may be involved in the regulation of citrus flowering.Through yeast double-hybrid,Pull-down and CO-IP experiments,it finds that there is no interaction between CcLIP2A and PtFT protein.Therefore,CcLIP2A does not participate in the flower formation pathway by interacting with FT protein,and its specific mechanism needs further exploration.In addition,the CcLIP2A may play a role in the process of citrus resistance to cold stress.