Cloning, Expression And Function Analysis Of GbCO And GbMFT In Gossypium Barbadense L.

Flowering is not only an important physiological phenomenon of flowering plants but also a particularcharacter of crops. Flowering could both influence the reproductive development and biomass of crops,playing a profound effect on the yield and quality of crops, which has been received many concerns all thetime. Recently, many key genes in control of flowering time passway were identified and their functionwere intensively explored, which contributing to revealing the mystery of flowering and moleculemechansim in plants a lot.Object: This project was focused on mining and function analysis of flowering related gene in cotten(Gossypium barbadense L.). CONSTANS (CO) was an important gene involved in the photoperiod pathwaycontrolling plant flowering time. MOTHER OF FT AND TFL11(MFT1) and MFT2are homolog ofFLOWERING LOCUS T (FT) which encode florigen in plants acting as an integrator of flowering passway.We aimed to clone these homologs in cotton and explore their possible roles in cotton flowering network,which will contribut to elucidating the molecular mechanism underlying flowering control in cotton infuture.Methods: GbCO, GbMFT1and GbMFT2homolog was isolated from cotton via RT-PCR and RACE; Wetransformed p35S:GbMFT1, p35S:GbMFT2, p35S:GbMFT1-GFPå'Œp35S:GbMFT2-GFP into Col-0andtobacco; The expression pattern of GbCO, GbMFT1and GbMFT2was studied using semi-quantitativeRT-PCR and fluorescence quantitative RT-PCR;Results:(1) A CONSTANS homolog was isolated from the fiber of9DPA (days post-anthesis) ofGossypium barbadense L. cultivar Xinhai14, which was designated as GbCO. The open reading frame(ORF) of GbCO is1008bp and encoded a putative protein of335amino acids. Similarity comparisonshowed that the amino acid sequence only shared78.2%identity with GhCO (GenBank: HM006910).Bioinformatics analysis indicated that the identities of the GbCO proteins were very high with Ricinuscommunnis protein RcCO, and Mangifera indica MaCO. qRT-PCR analysis demonstrated that GbCOexpressed in root, stem, leaves, and other organs, with the highest level in leaves than in other tissues.GbCO was also expressed in initiation and elongation stag of fiber in cotton, and arrived at higher level atthe-1DPA and5DPA. The expression pattern of GbCO was influenced by daily oscillation, with thehigher expression level in dark than that in light period.(2) One MFT-like homolog was isolated fromGossypium barbadense L. CV. Xinhai14, which was designated GbMFT1gene (GenBank:KC513744).GbMFT1ORF is528bp in length, encoding a putative protein of175amino acids. GbMFT1gene has a phosphatidylethanolamin-bing protein (PEBP) conserved motif. Liking almost all MFT-likeproteins, GbMFT1protein has a conserved proline residue near the C-terminus. Phylogenetic analysisrevealed that GbMFT1showed closer kinship with that of VvMFT and SELF PRUNING2G (SP2G),indicating that they belong to the same evolutionary branch. Expression analysis by qRT-PCR indicatedthat GbMFT1displayed a much broader expression range, but appears to reach its maximum expression in petal. GbMFT1was also expressed in different development stages of cotton fibres, but strongly expressedin the2DPA ovules and9DPA fibres. Expression analysis by semi-quantitative method indicated thatGbMFT1had a high expression level at the stags of germinated seed. No significant difference ofexpression in germinating seeds treated with different concentrations of ABA was detected, whichindicating GbMFT1expression was not regulated by ABA. Over-expression of GbMFT1in tabacco usingthe CaMV35S promoter resulted in significant accelerated flowering compared with wild-type plants. Thefusion protein of GbMFT1-GFP was located in both the plasma membrane and nucleus.(3) AnotherMFT-like homolog was isolated from Gossypium barbadense L. CV. Xinhai14, which was designatedGbMFT2gene. The GbMFT2ORF is519bp in length, encoding a putative protein of172amino acids.GbMFT2gene has a conserved PEBP motif. GbMFT2protein has also a conserved proline residue near theC-terminus. Phylogenetic analysis revealed that GbMFT2showed closer kinship with that of AtMFT,indicating that they belong to the same evolutionary branch. Expression analysis indicated that GbMFT2display a lower expression in cotton tissues, but appears to reach its maximum expression in ovules.GbMFT2was also expressed in different development stages of cotton ovules, but strongly expressed in the25DPA ovules. The expression levels of GbMFT2in germinating seeds were gradually elevated along withincreased concentrations of ABA treatment, and GbMFT2was markedly upregulated in just-germinatedseeds compared with geminating seeds treated with high concentrations of ABA. Overexpression ofGbMFT2in tabacco using the CaMV35S promoter resulted in accelerated flowering compared withwild-type plants. GbMFT2protein was also located in both the plasma membrane and nucleus.Conclusion: GbCO, GbMFT1, and GbMFT2may play important roles in regulating flowering time duringthe process of flower development in Gossypium barbadense L. Moreover, GbMFT2aslo plays a key rolein the regulation of germination in cotten.