Cloning And Expression Analysis Of The Genes Associated With Cytosine Methylation And Sumoylation(Litchi Chinensis Sonn.)

In this study,full-length cDNA sequence of two cytosine methyltransferase genes(named LcCMT3 and LcDRM2),partial cDNA sequence of cytosine methyltransferase 1 gene(named LcMET1)and four full-length cDNA sequence of SUMOylation related genes(named LcSAE2,LcSCE1,LcSUMO1-a and LcSUMO1-b)were obtained from the leaves of Litchi chinensis cv.Yuanhong by using RT-PCR and RACE methods.Then the obtained genes were subjected to bioinformatic ananlysis.The expression levels in different tissues and organs,and in kinds of cotyledon embryos(induced from Yuanhong Litchi anther)were analyzed.In addition,the gemome DNA methylation levels in the materials we used were investigated by using Methylation Sensitive Amplification Polymorphism(MSAP).Results were listed as follows:1 Cloning of Litchi DNA methyltransferase genes.Three DNA methyltransferase genes,i.e.LcMET1(Genbank No.KY780308),LcCMT3(Genbank No.KX886206)and LcDRM2(Genbank No.KY780307),were cloned from litch leaves via homologous cloning.Blast alignment analysis showed that LcMET1(partial)shares high similarity with MET1 gene from other species.The proteins encoded by LcCMT3 gene and LcDRM2 gene were further analyzed and results showed that the LcCMT3 could encode a protein with 877 amino acids,and the LcDRM2 could encode a protein with 631 amino acids.Bioinformatic analysis also found that both LcCMT1 and LcDRM2 werenucleus located hybrophilic proteins with DNA methyltransferase function domain but without signal peptide and coiled domain.2 Cloning of Litchi SUMOylation reated gene from litchi.Four SUMOylation reated genes,i.e.LcSAE2(Genbank No.KX886205),LcSCE1(Genbank No.KY170859).LcSUMO1-a(Genbank No.KY780305)and LcSUMO1-b(Genbank No.KY780306),were cloned from litch leaves via homologous cloning.The proteins encoded by LcSAE2 gene,LcSCE1 gene LcSUMO1-a gene and LcSUMO1-b gene were further analyzed and results showed that the LcSAE2 could encode a protein with 643 amino acids,LcSCE1 could encode a protein with 160 amino acids,LcSUMO1-a could encode a protein with 105 amino acids,and the LcSUMO1-b could encode a protein with 100 amino acids.Bioinformatic analysis also found that both LcSAE2,LcSCE1,LcSUMO1-a and LcSUMO1-b.were nucleus located hybrophilic proteins with SUMOylation reated function domain but without signal peptide and coiled domain.3 Expression analysis of Litchi Cytosine Methylation and Sumoylation related genes during different Somatic Embryogenesis and different part of litchi.Quantitative Real-time PCR result showed that LcSAE2,LcSCEl and LcSUMO1-b were highly expressed in the smallest cotyledon embryos(type V),suggesting thatSUMO1-b modifications could inhibit somatic embryos development.The expression levels of LcMET1,LcCMT3 and LcDRM2 in type Ⅲ and type Ⅸ cotyledon embryos(both with longer embryos and top of cotyledons opening)were all very low.It is presumed that LcMETl,LcCMT3 and LcDRM2 could inhibit the development of somatic embryos,but the specific mechanism requires further investigation.Quantitative Real-time PCR result showed that the expression levels of LcMET1,LcCMT3,LcDRM2,LcSAE2 and LcSCEl in the aborted seeds were all significantly higher than those in the normal seeds,suggesting that DNA methylation and SUMOylation may play an important roles during litchi embryonic development and the differential expression of these DNA methylation and SUMOylation related genes may contribute to the formation of the aborted seeds.Moreover,SUMOylation related genes showed high expression in fast-growing tissues or organs,which indicated that SUMOylation may contribute to the growth and development of litchi.Moreover,the expression of LcMET1,LcCMT3,LcDRM2,LcSAE2,LcSCE1,LcSUMO1-a and LcSUMO1-b genes showed significant differences between female and male flowers.Whether DNA methylation and SUMOylation function in the flower sex determination need to be further investigated.4 Optimization of MSAP analysis system of Litchi and DNA methylation analysis during different part of litchi.Genomic DNA of "Yuanhong" litchi was used as template for the MSAP system optimization.It was shown that The concentration of primer was 0.4mM·L-1.By comparing with some other plant species,higher DNA methylation level was found in all litchi tissues and organs.And its full methylation level is slightly higher than the level of semi-methylation,indicating that the phenomenon of single-stranded external cytosine methylation or internal and external cytosine methylation in "Yuanhong" litchi was less than that of double-stranded cytosine methylation.No significant difference was found among the expanding seed,aborted seed and normal seed.Although the overall methylation level of the expanding fruit seed is slightly lower than that of the aborted seed and normal seed.However,the total methylation level in the aborted seed(27.41%)was significantly lower than that in the normal seed(36.57%),and the hemi-methylation level of the normal seed(24.63%)was significantly lower than that of the aborted seed(32.59%).It is speculated that genomic DNA methylation plays an important role in the development of the seed during the development of the expanded seed and it may function in the formation of aborted seed.DNA methylation level changes,as an important regulation way of gene transcription,must be play an important role in the transcription of certain genes in the embryonic development of litchi.The changes of methylation status in the gene and its promoter could influence the gene’s expression,affect the embryo development and determine the formation of aborted seed or normal seed.The results generated in this study could provide basis for the understanding of the formation mechanism of absorted seed in litchi.