Isolation And Expression Analysis Of DNA Methyltransferase Genes In Strawberry And Apple

Along with the development of plant micro-propagation technique and the application of strawberry micropropagated plants in production,epigenetic variation in strawberry micropropagated plants attract researchers'attention.Methylation of cytosine residues,the most common epigenetic modification of DNA in higher eukaryotes,has been implicated in many biological processes as a regulator for plant growth,development and evolution. Cytosine methylation is catalyzed by DNA cytosine 5-methyltransferase(DMTs).Here,two families(MET1 and DRM) of DNA methyltransferase genes,most likely function as maintenance and de novo methyltransferase,respectively,were firstly isolated from strawberry.Expression profiles of strawberry DMTs were compared among micropropagated plants and their progeny by real-time quantitative PCR.Moreover,the effects of strawberry viruses on the transcript level of DMTs were analyzed.The expression profiles in diploid-and tetraploid apple plants were analyzed by semi-quantitative RT-PCR method.The main results were as follows.1.One and two MET1 gene fragments were isolated from strawberry and apple, respectively,using degenerate PCR amplification.The fragment was 443 bp and contained two intact motifs(ⅣandⅥ) and two incomplete motifs(ⅡandⅦ).Two DRM gene fragments respectively,were isolated from strawberry and apple.The fragment was 323 bp and contained two intact motifs(ⅠandⅢ) and two incomplete motifs(ⅩandⅣ).2.The full-length gene sequences were approached by chromosome walking.For MET1 gene,four steps including one step downstream and three successive steps upstream amplifications generated fragments of 1634,1812,1308 and 2786 bp,with overlapping sequences of 57,76,87 and 195 bp to establish a contig sequence of a maximum size of 8171 bp.For DRM gene,five steps including one step downstream and four successive steps upstream amplifications generated fragments of 1405,1835,2354,2142 and 1847 bp,with overlapping sequences of 131,75,64,250 and 372 bp to establish a contig sequence of a maximum size of 9014 bp.3.Three primer pairs which were designed according to the full-length gene sequences obtained from chromosome walking were used to obtain full-length cDNA sequence of MET1 gene combined with 5'and 3' RACE methodologies.Two contig cDNA sequences of 5032 and 5002 bp were determined,which encoded the predicted protein of 1565 and 1557 amino acid residues with calculated mass of 173.946 and 173.064 kDa.The gene products were designated as Fragaria×ananassa MET1a(FaMET1a) and Fragaria×ananassa MET1b (FaMET1b),respectively.Two BAH(Bromo-adjacent homology) and eight conserved motifs were predicted in FaMET1 genes.The two fragments shared 98.63%nucleotide and 98.72% amino acid identity.FaMET1,PpMET1,PtDMT901 and PtDMT901 fell in the same clade of group 1 in phylogenetic analysis.For DRM gene,full-length cDNA sequences were obtained by 5' and 3' RACE methodologies.Three contig cDNA sequences of 2273,2282 and 2288 bp were determined,which encoded a predicted protein of 596 amino acid residues with calculated mass of 67.304,67.325 and 67.304 kDa.The gene products were designated as Fragaria×ananassa DRMa(FaDRMa),Fragaria×ananassa DRMb(FaDRMb) and Fragaria×ananassa DRMc(FaDRMc),respectively.The UBA(ubiquitin-associated) domains and seven conserved motifs were predicted in FaDRM genes.The nucleotide identity between FaDRMa and FaDRMb,FaDRMa and FaDRMc,FaDRMb and FaDRMc were 94.78%,97.40%and 97.17%,respectively and amino acid identity between FaDRMa and FaDRMc,FaDRMa and FaDRMb were 100%and 98.49%.FaDRM,NtDRM1 and SlDRM5 fell in the same clade of group 1 in phylogenetic analysis.4.In order to identify introns,sequence alignment was performed on genomic DNA and cDNA.Two introns were scanned in FaMET1 genes and ten introns with different sizes were dispersed in FaDRM genes which harboured the GT-AG consensus.Sequence analysis revealed that there were alternately spliced transcripts in FaDRM genes,which might be classified into two types i.e.alternative 3' splicing and intron retention.5.The simple and rapid technique system had been established with total nucleic acid as the template for simultaneous detection of strawberry RNA and DNA viruses by RT-PCR.The micropropagated plants from strawberry cultivars'Toyonoka' and'Allstar' were identified to confirm the types of virus.The micropropagated plants with/without vitus and the first runner generation of micropropagated plants with/without virus of'Toyonoka' and'Allstar' were used for studying expression profiles of DMTs by real-time quantitative PCR based on Taqman probe.The results indicated that virus could up-regulate DMTs expression,and the expression of DNA methyltransferase in the first runner generation of micropropagated plants without vitus was higher than that in plants with virus.6.Expression of strawberry DMTs was compared among micropropagated plants and their progeny of'Toyonoka' by real-time quantitative PCR based on Taqman probe.The results indicated that a consistent increase in transcript levels of FaMET1 and FaDRM was found.The transcriptional expression of both FaMET1 and FaDRM genes was down-regulated in three types of micropropagated plants,and the expression level of DMTs was recovered in the first and second runner generations of micropropagated plants.7.Semi-quantitative RT-PCR method was used to investigate the expression of MET1 and DRM genes in diploid and colchicine- induced tetraploid plants of'Hanfu' apple.The results indicated that MET1 gene expression level in the young leaves from diploid plants was slight higher than that from tetraploid plants(0.85/0.74),and very slight difference in the MET1 gene expression level was observed between the mature leaves both from diploid and tetraploid plants.But MET1 gene expression level in the young leaves was significantly higher than that in mature leaves both for diploid and tetraploid plants.The differences of DRM gene expression level between young and mature leaves and between diploid and tetraploid plants were very slight.8.The regulatory regions of 669 bp and 901 bp upstream of FaMET1 and FaDRM genes respectively were cloned into the pCAMBIA1301 binary vector by replacing the CaMV 35S promoter giving rise to plasmids FMpro::GUS and FDpro::GUS.An Agrobacterium strain carrying the recombined plasmid was injected into strawberry fruits.The transient expression of GUS gene indicated that the cloned fragments of strawberry DNA methyltransferase genes had promoter activity.