The DNA Methylation And Expression Analysis Of BrVIN3 Gene Family During The Course Of Vernalization In Brassica Rapa

Plants tolerate low temperature in winter and flowering in next spring this phenomenon is called vernalization.In Arabidopsisthaliana,vernalization can produce the transcript abundance of VERNALIZATION INSENSITIVE 3(VIN3).During vernalization VIN3 and its homologues can work together withPolycomb Repressive Complex 2(PRC2)to repressive the expression of FLOWERING LOCUSC(FLC)which is acting as a floral repressor.In Brassica rapa,BrVIL1and BrVIL2 are the homologues of BrVIN3.In Arabidopsisthaliana,VIN3 mRNA is expressed only during thevernalizing cold.The mRNA of VIL1lis expressed during and after vernalization,this phenomenon suggest VIL1 may have a function in the continue repression of FLC and FLM after vernalization.And the expression of VIL1 is produced constitutively.The expression of VIL2 can be induced after vernalizing 30 days,and after vernalization the expression of VIL2 is reduced.DNA methylation is a kind of epigenetic mark,which can affect gene expression but don't change its sequence.Generally speaking,DNAmethylation of gene promoter suppresses gene expression,however the relationship between DNA methylation of gene bodyand gene expression depends on species.In this study we annlyzed the expression and methylation patterns of VIN3 gene family in three different genotypic Bra.rapas,and we cloned BcVIL1 gene in non-heading Chinese cabbage,and had a preliminary analysis of its structure and function.The main results are as follows:1.We cloned BcVIL1 gene in non-heading Chinese cabbage and analyzed the expression patterns of BcVILl in'PC-175' and 'CX-49' during the course of vernalization.So we had a preliminary analysis of its structure and function.The sequences of BcVIL1 was consistent in two genotypic non-heading Chinese cabbage,and BcVIL1 containing a 1020 bp open reading frame encoded 339 amino acid residues.The bioinformatics analysis show that homology between Brassica campestris ssp.chinensis Makino and Brassica rapa ssp.pekinensis was very high at 99%.In 'PC-175' the BcVILl could be induced by 7 days of vernalizaingand the expression of BcVILl was in high level during and after the course of vernalization.However in 'CX-49' the expression of BcVIL1 had no obvious different compering with non-vernalization condition.Indicating that the expression of BcVILl was different in two genotypes.The BcVILl gene was verified to be located in cell nucleus and membrane by sub-cellular localization.2.Using qRT-PCR we analyzed the expression patterns of BrVIN3 gene family in'CX-49','CC-072' and 'PC-Glu' in non-vernalization(NV),vernalizing 14 days(14V),vernalizing 21 days(21V),vernalizing 28 days(28V),after vernalizing 28 days transfer to normal condition for 7 days(28VT7).And we found that the expression of BrVIN3 A03 and BrVIL2_A03 presented a significant change during the course of vernalization.So we produced DNA methylation sequences of BrVIN3 A03 and BrVIL2_A03 by Bisulphite Sequencing.And we found that the methylation levels of BrVIN3_A03 and BrVIL2 A03 promoter was very low in three materails.In 'CX-49',there was a positive connection between DNA methylatedof BrVIN3_A03gene body and its expression during the course of vernalization.In 'CC-072',there was also a positive connection between DNA methylatedof BrVIL2_A03 gene body and its expression during the course of vernalization.However in'PC-Glu',the DNA methylatedof BrVIN3_A03 and BrVIL2_A03 gene body was in low levels and had no connection with their expression during vernlization.These results suggest that the DNA methylatedof BrVIN3 A03 and BrVIL2_A03 can promote their expression,but only in specific specific.We also found two significant methylation sites of BrVIL2_A03 gene body were both in the FN3 domain.This result may suggest that the DNA methylation of BrVIL2 A03 can affect its function.