Cloning And Functional Identification Of AcFTL1 And AcFTL2 Genes From Pineapple(Ananas Comosus)

Pineapple is one of the three tropical herbal fruit trees in the world.Because of its unique flavor and nutritional value,it has high commercial value.In recent years,China's pineapple industry has no competitive advantage in domestic and foreign markets.Flowering is a turning point from vegetative growth to reproductive growth,which has a profuond impact on fruit formation,fruit yield and quality.Ethylene-induced flowering of pineapple is a conventional cultivation technique,but its molecular mechanism is still in the initial stage.FT is an important regulator that integrates multiple pathways of flowering induction signals and transmits them to downstream floral meristem to determine genes,so it has important research value.Studies have shown that pineapple AcFT gene expression is regulated by ethylene,and can promote Arabidopsis flowering time to advance about 6.8 days and increase the number of floret.In this study,two new FT homologous genes,AcFTL1 and AcFTL2,were cloned from "MD-2" pineapple,and the response characteristics of AcFTL1,AcFTL2 and control genes(AcFT)to exogenous ethylene at low concentration(600 ppm)and high concentration(1200 ppm)were detected by qRT-PCR.Then,the bioinformatics was used to analysize the physical and chemical properties,coserved domain,homology,phylogenetic tree and signal peptide,etc.of AcFTL1 and AcFTL2 proteins.The over-expression vectors of 35S:: AcFTL1 and35S::AcFTL2 were constructed and transformed into wild-type Arabidopsis thaliana by agrobacterium-mediated method for functional identification of AcFTL1 and AcFTL2 genes.Finally,the interactions among AcFTL1,AcFTL2,AcFDL1,AcFDL2 and Ac14-3-3L proteins were detected by yeast two-hybrid technique.The results showed that:(1)AcFTL1,AcFTL2 and AcFT genes could all be induced by low and high concentrations of ethylene,and they were more sensitive to high concentrations of ethylene;however,AcFTL1 and AcFTL2 were much more sensitive to low and high concentrations of ethylene than AcFTL1,especially AcFTL1,which indicated that AcFTL1 and AcFTL2 were more likely than AcFT to be key genes in the process of ethylene-induced flowering of "MD-2" pineapple.(2)The proximal N-terminal of AcFTL1 and AcFTL2 proteins each contain a signal peptide,and the two proteins are closely related to the flowering-promoting members(FT and TSF)of the FT/TFL subfamily of Arabidopsis thaliana in phylogenetic tree,suggesting that they may have transport function in cells or tissues of pineapple,and may also be related to promoting pineapple flowering.(3)Overexpression of AcFTL1 gene can advance Arabidopsis thaliana flowering time by about 11-12 days,and can also make lotus leaf of Arabidopsis thaliana wrinkle(especially tip position of leaf);however,overexpression of AcFTL2 gene has no any effect on Arabidopsis thaliana flowering period.(4)AcFTL1 protein can interact with AcFDL1 and AcFDL2 proteins respectively,but can not interact directly with Ac14-3-3L protein;and Ac14-3-3L protein can interact with AcFDL1 and AcFDL2 respectively;however,AcFTL2 protein can not interact with anyone of AcFDL1,AcFDL2 and Ac14-3-3L proteins.This indicate that AcFTL1,AcFDL1,AcFDL2 and Ac14-3-3L proteins may have capacity to form AcFTL1-AcFDL1-Ac14-3-3L or AcFTL1-AcFDL2-Ac14-3-3Lprotein complexes in pineapple and then regulate the expression of downstream genes to promote pineapple flowering.