Cloning And Functional Analysis Of Galactinol And Raffinose Synthase Genes In Prunus Mume

Mei(Prunus mume Sieb.et Zucc.)originated in the South of China,but low temperature in winter limits its cultivation in the North of China.The accumulation of raffinose family oligosaccharides(RFOs)could improve the resistance and adaptation to stress in plants,which is an important osmotic adjustment substance in plant cells.It is synthesized by adding galactosyls to sucrose gradually,and inositol galactoside synthase(GolS)and raffinose synthase(RS)are the key enzymes in the process.In order to identify the function of PmGolS and PmRS genes in response to abiotic stress,five PmGols(Pm003196,Pm006697,Pm008430,Pm012900,Pm020004)and two PmRS(Pm027594,Pm025896)from P.mume ‘Xue Mei' were isolated in our research,whose overexpression vectors were constructed,respectively.qRT-PCR was used to analyse their expression patterns under circadian rhythm and abiotic stresses.Meanwhile the genes were transferred into Arabidopsis to identify their function under abiotic stresses.In addition,they were transformed into Prunus mume,and transgenic seedlings were obtained with roots.The main results were as follows:1.Cloning and amino acid sequence analysis of PmGolS and PmRS genesFive PmGolS and two PmRS genes were cloned according to the data of gene expression profiling at 4 ? and the genome of Prunus mume.Phylogenetic analysis of GolS showed that the similarity of GolS homologous proteins among different species was higher than that of different GolS subtypes in the same species,indicating that species evolution may occur after intracellular Gol S gene sequence replication events.Phylogenetic analysis of RS was consistent with GolS.The analysis of amino acid sequence revealed that the amino acid sequences of PmGolS were highly conserved and all amino acid sequences has a manganese binding elements DXD at position 130,a serine phosphorylation sites at position 270,and a characteristic hydrophobic pentapeptide(APSAA)at carboxyl terminal.While the amino acid sequence of PmRS varied,but they both have a KXD and a RXXXD conservative catalytic domains.2.The expression patterns of PmGolS and PmRS genes under circadian rhythm and abiotic stressesUsed young leaves of P.mume ‘Xue Mei' as material,qRT-PCR was hired to analyse the expression patterns of five PmGolS and two PmRS genes under circadian rhythm andabiotic stresses include low temperature,drought,high salt and ABA.The results indicated that Pm003196 had obvious circadian rhythm expression pattern,and the other six genes had no obvious circadian rhythm expression;All genes didn't respond to ABA stress,but had different responses to the other three abiotic stresses: Pm003196,Pm006697 and Pm025896 responsive to low temperature stress,Pm008430,Pm027594,and Pm025896 responsive to NaCl stress,Pm003196,Pm008430,Pm025896 and Pm027594 in answer to mannitol stress,not response of Pm027594 to low temperature stress completely,low expression level of Pm012900 and Pm020004 in all stress conditions.3.The subcellular localization of Pm Gol SThree genes(Pm003196,Pm008430 and Pm006697)in the PmGolS family were cloned,and the corresponding YFP fusion expression vector was constructed for subcellular localization.The results showed that the three proteins are located in the nucleus,membrane and cytoplasm.4.Genetic transformation of Arabidopsis thaliana by PmGol S and PmRS genes and its abiotic stress resistanceFive PmGolS and two Pm RS genes were cloned and the overexpression vectors were constructed,respectively.We obtained the Arabidopsis thaliana transformed lines of all genes.T1 transgenic Arabidopsis plants were analyzed by RT-PCR,which displayed that Arabidopsis lines with high expression levels of all genes except Pm020004 were obtained.The low temperature freezing treatment and relative electrical conductivity(REC)determination among the T2 transgenic Arabidopsis lines and WT were conducted.The results showed that the survival rate and REC of the transgenic Arabidopsis lines overexpressing PmGol S were not significantly different from wild-type lines;However,the survival rate of the transgenic Arabidopsis lines which overexpressed PmRS were higher than that of wild-type Arabidopsis,and the REC were lower than that of wild-type Arabidopsis;In addition,the germination rate of Pm003196 overexpressing Arabidopsis seedlings at 400 mM mannitol stress was higher than that of WT.5.Genetic transformation of Prunus mume transferred by PmGolS genesAgrobacterium-mediated transformation of PmGolS was carried out with immature embryo cotyledons as explants.262 explants of immature embryo cotyledons wereinoculated,and 24 resistant buds were obtained.After transferred to elongation medium,the length of 15 resistant buds reached 2 cm.The PCR test showed that there are 8positive plants.One shoot over-expressing Pm008430 rooted in the medium after subcultured for 120 d to 150 d.