FLOWERING LOCUS C1 Confers Resistance To Bolting In WuCai (Brassica Campestris L.) With A Single Point Mutant

The flowering,signal of transition from vegetative to reproductive growth,is crucial in the life history of higher plants.It is responding to environmental and endogenous signals,and the proper time of flowering is crucial for crops to adapt to the local climatological condition.However,there are many crops early-flowering due to environmental conditions and heredity in production,which seriously affect crop yield and quality.Wucai?Brassica campestris L.?is a variant of cruciferous cabbage,known for its high vitamin C content and extremely resistant to low temperatures.As a widely cultivated vegetable distributing in the Yangtze–Huaihe river basin,it plagued the majority of producers due to early-flowering.Breeding the cultivar resistant to bolting is an effective way to solve this problem.Grasping the molecular mechanism of flowering is significant.There are several pathways controlling flowering time in Brassica,but most pathways share FLOWERING LOCUS C?FLC?and SHORT VEGETATIVE PAHSE?SVP?as a target.FLC encodes a MADS-box protein and binds to downstream gene promoters.It is a floral repressor.FLC can form protein complexes with SVP,together affect the expression of FLOWERING LOCUS T?FT?and SUPPERSS OF OVEREXPRESSION CONSTANS 1?SOC1?and control the flowering of crops.In this experiment,the different time of flowering were analyzed by cloning the FLC1 of two WuCai genotypes?W-1,lately-bolting;W-2,early-bolting?.The main results are as follows:1.The complete encoding sequence of FLC1 gene and SVP gene was successfully obtained by homologous cloning from two WuCai genotypes.The BLAST analysis showed that the FLC1 gene had the highest similarity with Brassica napus and was named BcFLC1.The two genes were 591 bp and encoded 196 amino acid residues.The protein that BcFLC1encoding was a MADS-box and a K-Box;there are two base mutations in the BcFLC1 gene,and the mutation at the latter base causes the mutation of 174th amino acid.The SVP gene was 726 bp and encoded 241 amino acid residues.2.The online prediction results showed that the BcFLC1 gene was localized in the nucleus.meantime,the pBI121-35S::BcFLC1-GFP expression vector was successfully constructed,and the vector was transformed to onion epidermal cells with the Agrobacterium tumefaciens EHA105,and subcellular localization confirmed that BcFLC1 was localized in the nucleus.3.The expression patterns of BcFLC1,SVP,SOC1,and FT in WuCai were analyzed by quantitative Real-time PCR.There was no significant difference in relative expression levels of FLC1 and SVP between the two genotypes in the same period,but SOC1 and FT differs greatly between the two genotypes,and the SOC1 and FT expression of the W-1 is significantly lower than that of the W-2.As the plant grows,FLC1 and SVP showed a downward trend in both genotypes,and FT and SOC1 showed an upward trend.At flowering period,FLC1 and SVP were highly expressed in roots and stem tissues,and extremely low in leaves,flowers and buds.FT and SOC1 genes were expressed in roots at low levels and were highly expressed in flower organs.4.The yeast two-hybrid system was used to analyze the effect of interaction between BcFLC1 and SVP,the BcFLC1 was inserted into the pGBKT7 vector and transformed into the Y2H Gold yeast strain.The SVP was inserted into the pGADT7 vector and transformed into Y187 yeast strain.There was no autoactivation and toxicity.Yeast mating results showed that the point mutations did not completely destroy the interaction between BcFLC1 and SVP,but mutations led to a decrease in interaction between the two genes.5.In order to analyze the effect of the decrease of interaction on the flowering,the overexpression vector pBI121-35S::BcFLC1 was constructed.It was transformed into Agrobacterium tumefaciens GV3101 and successfully transformed into Arabidopsis thaliana by floral dip method.Three T0 plants with 35S::BcFLC1-1 transgene and two T0plants with 35S::BcFLC1-2 were screened.After screening,35S::BcFLC1-1 lines were obtained in T₃ generation for a total of 29 plants,A total of 26 plants were sampled in the35S::BcFLC1-2 strain.And the bolting statistics showed that the 35S::BcFLC1-1 lines had late bolting and increased leaf number.The results of quantitative Real-time PCR showed that the relative expression level of FT and SOC1 was lower in the transgenic plants compared with wild-type plants,and the relative expression level of FT and SOC1 in35S::BcFLC1-1 transgenic lines were significantly decreased.Therefore,point mutations in the BcFLC1 gene can affect its interaction with the SVP gene,which in turn affects the expression of the downstream floral integrators integration FT and SOC1 genes,and eventually leads to late bolting of the plant.