Cloning Of The FT Genes From Safflower And Its Involvement In Flower Formation And Flavonoid Synthesis

Safflower(Carthamustin ctorius L.)is a traditional medicinal and oil plant in China.Flavonoids are the main effective components of Carthamus tinctorius petals and have a variety of pharmacological effects as an important factor affecting its economic value.Carthamus tinctorius,as a plant that likes dry environment,is restricted in the humid and rainy season in summer,resulting in damage to its yield and quality,and advancing or delaying the flowering time of safflower will solve this problem to a certain extent.At present,floral factor(FLOWERING LOCUS T)genes have been cloned and their functions have been studied in many plants,but they have not been reported in safflower.Therefore,it is important to explore the relationship between flowering and flavonoid synthesis.In this study,three FT genes of Carthamus tinctorius were screened and identified from PEBP gene family and analyzed by bioinformatics.Through heterologous expression in Arabidopsis thaliana and transient overexpression of CtFTs genes in safflower,the relative expression of related genes and the content of total flavonoids were detected,and the interaction of related proteins was verified to explore the function of CtFTs.The main results are as follows:1. Based on the genome sequencing of safflower(Jihong No.1),seven PEBP genes were screened.The phylogenetic tree was constructed with the amino acid sequences of Arabidopsis thaliana and rice PEBP family.The results showed that three CtFT were clustered in the FT-like subfamily,three CtTFL1 were clustered in the TFL1-like subfamily,and one CtMFT was clustered in the MFT-like subfamily.Protein secondary structure analysis showed that the full-length 174～176 aa of FT-like subfamily protein,the theoretical isoelectric point is 6.29～7.75,the molecular weight was 19681.24～19933.46 Da;TFL1-like subfamily proteins with a total length of 152～212 aa,the theoretical isoelectric point is 4.76～9.16,the molecular weight was17259.85～23811.78 Da;The full length of amino acid sequence of CtMFT is 151 aa,the theoretical isoelectric point is 9.55,the molecular weight was 16987.44 Da.Except for CtTFL1-2,the other 6 PEBP proteins are unstable,and all CtPEBP proteins are hydrophilic proteins.Subcellular localization predicts that CtFT1,CtFT2,CtFT2,CtTFL1-1 and CtMFT are located in the cytoplasm,while CtTFL1-2 and CtTFL1-3 are located in microbodies.Gene structure and motif analysis showed that CtFT1,CtFT2,CtFT3 and CtTFL1-3 had 4 exons and 3 introns,while CtTFL1-1,CtTFL1-2 and CtMFT had 3 exons and 2 introns,three CtFTs have the same motif.Promoter element analysis showed that the functions of cis-acting elements in the upstream promoter of CtFTs coding region were mostly concentrated in response to external stress,and the more concentrated ones were those involved in light response.Because CtFT3 had four cis-acting elements in response to abscisic acid,it was speculated that it might be more special than the other two genes in response to external hormones.The results of multiple sequence alignment with Arabidopsis thaliana,Glycine max and Oryza sativa L.FT genes showed that except for the first aspartic acid(D)mutation to asparagine(N)in the D-P-D-x-P conserved domain of CtFT2,CtFTs had PEBP family conserved domains D-P-D-x-P,G-x-H-R,and tyrosine(Y),the 85 th key amino acid residue that distinguishes FT-like from TFL1-like subfamily.Glutamine(Q),the key amino acid residue at the 144 th position,has a conserved domain Segment B(LGRQTVYAPGWRQN)regulating floral formation.The first amino acid in the triad of YN triad in safflower FT proteins is alanine(A),which may imply that CtFTs may have different functions.Conserved domain analysis showed that safflower CtFTs proteins had typical PEBP conserved domain.The coding region sequences of CtFT1,CtFT2 and CtFT3 genes were cloned from safflower leaf c DNA.2. The plant expression vectors of pCAMBIA3301-CtFT1,pCAMBIA3301-CtFT2 and pCAMBIA3301-CtFT3 were constructed.After Agrobacterium tumefaciens transformation,Arabidopsis thaliana was infected by flower soaking.After resistance screening and PCR detection,T3 generations of transgenic Arabidopsis thaliana overexpressing CtFT1,CtFT2 and CtFT3 were obtained.Compared with WT and ft mutants Arabidopsis thaliana,CtFT2 can promote Arabidopsis early flowering,while CtFT1 and CtFT3 delay Arabidopsis flowering to a similar extent,but earlier than ft mutants.The number of rosette leaves of CtFT2 transgenic lines was similar to that of WT,while that of CtFT1 and CtFT3 lines was slightly more than that of WT,but the number of rosette leaves of ft Arabidopsis was significantly higher than that of the other four lines.The main stem height of CtFT2 transgenic Arabidopsis thaliana was slightly lower than that of WT,CtFT1,CtFT3 and ft lines at the first flowering,and higher than WT,followed by ft,CtFT1 and CtFT3 from high to low.There is a protein interaction between CtFTs and CtGRF5,a constituent protein of floral activation complex.Overexpression of CtFT1 and CtFT2 genes had no significant effect on flavonoid biosynthesis in Arabidopsis,but overexpression of CtFT3 promoted flavonoid biosynthesis in Arabidopsis thaliana.3. The expression patterns of CtFTs in Carthamus tinctorius under different light conditions showed that the expression level of CtFTs in long days was higher than that in short days,the expression pattern of CtFT1 was highly consistent with CtFT2,and the expression of CtFT3 was low and did not change significantly under long and short days.The expression vectors of p Green II62-SK-CtFT1,p Green II62-SK-CtFT1 and p Green II62-SK-CtFT1 were constructed.After Agrobacterium tumefaciens transformation,safflower leaves were injected with CtFTs genes for transient overexpression.20 days later,compared with the empty vector safflower injected with p Green II62-SK,CtFT1 promoted early flowering of safflower,while CtFT2 and CtFT3 delayed the flowering of safflower.Transient overexpression of CtFTs genes increased the content of total flavonoids in safflower leaves and the expression of key enzyme genes in flavonoid biosynthesis pathway.