In Vitro Conservation Of Peach And Pear And Cloning Of SOD Genes From Peach

Both peach (Prunus persica Linn.) and pear (Pyrus spp.) were important woody fruit trees in China. The pear and peach germplasm resources faced crisis of loss, so it was very important to reinforce the research of germplasm resources conservation. As the first line of defense against ROS, superoxide dismutase (SOD) plays a key role in the process of in vitro conservation. In view of this, peach and pear were used as the materials on the researches of in vitro conservation, and further gene cloning of SODs from peach embryogenic callus (EC).The results provided better knowledge about the mechanism of funcction of SODs in vitro conservation. The main results were as follows:1Study on in vitro conservation of peach1.1Study on in vitro conservation of peach ECIn this experiment the ’Premier’ Honey Peach EC were used as materials for the studies on the effects of different concentrations of sugar, PP333, mannitol and nositol on in vitro conservation of peach EC. The results showed that the best concentration of sugar was40g/L, the best concentration of PP333was10mg/L, the best concentration of mannitol was20g/L, the best concentration of nositol was0.1g/L. Cultured on the MS+1.2mg/L2,4-D+20g/L mannitol+O.lg/L nositol+30g/L sucrose+6g/L agar medium, the subculture cycle of ’Premier’ Honey Peach EC could be prolonged to60d.1.2In vitro conservation of the plantlets from mature embryo culture of peachThe mature embryo of white peach was used as the material for in vitro culture and conservation. The results showed that the best proliferation medium was MS+1.0mg/L6-BA+0.0mg/L IBA+30g/L sucrose+6g/L agar, with the proliferation coefficient of3.45; the best medium for rooting was1/4MS+0.0mg/L IBA+0.4mg/L NAA+30g/L sucrose+6g/L agar, with the rooting rate of82.22%; the best rooting medium was1/2MS+0.5mg/L IBA+0.0mg/L NAA+30g/L sucrose+6g/L agar, with the average root number of5.96per plantlets. Adding3g/L mannitol to the medium was beneficial to a short-term conservation (<5months) but not beneficial a long-term conservation。2Study on in vitro conservation of pear2.1Establishment of the regeneration system of pearThe seeds were used as the material for establishing the plant regeneration system in the pear cultivar’Whangkumbe’.The results showed that the best medium for germination was1/2MS+0.4mg/L6-BA, with the germination percentage was93.02%; the best proliferation medium was MS+1.0mg/L6-BA+0.2mg/L IBA+0.2mg/L GA3, with the proliferation coefficient of4.72; the best medium for rooting was1/4MS+1.0mg/L NAA+1.0mg/L IBA+20g/L sucrose, with the rooting rate of65.79%; the best rooting medium was1/2MS+0.0mg/L NAA+1.0mg/L IBA+40g/L sucrose, with the average root number of3.96per plantlet.2.2Optimization of in vitro comservation system and in vitro conservation of the germplasm resources in pearsThe test-tube plantlets of pear ’Whangkumbe’ were used as the materials for in vitro conservation and screening the best conservation medium. The results showed that the best conservation medium was MS+0.5mg/L6-BA+0.1mg/L IBA+6.0g/L agar+30g/L sucrose+6mg/L PP333+5g/L mannitol.10accessions of pear germplasm resources were conserved in vitro using this conservation medium. The growth and survival rates of the pear germplasm resources were observed after in vitro conservation for6months, and the results showed that the survival rates of the wild species were higher than those of the cultivated species.3Cloning of SOD genes family from peach ECIn this experiment, on the basis of the research on in vitro conservation,22different mRNA transcripts from SOD gene family which included Fe-SOD, Mn-SOD and Cu/Zn-SOD genes were cloned from peach EC by RT-PCR combined with RACE,21of them were the full-length sequences. 3.1Cloning of Fe-SOD genes8different mRNA transcripts of Fe-SOD genes which were cloned from ’Premier’ Honey Peach EC were obtained in the experiment. The sequences of the full-length cDNA were from1220bp to1235bp. In order of the G+C content, it was:5’UTR>3’UTR> ORF. The sequence feature analysis showed that Fe-SOD gene existed2different transcription initiation sites (TSS) and3different polyadenylation sites. The results deduced that there existed alternative splicing in Fe-SOD genes.8different mRNA transcripts of Fe-SOD gene encoded2proteins.The2proteins were consisted of319and320amino acids, respectively. The bioinformatics analysis revealed that these2proteins had the similar physicochemical property. Both of the2proteins existed coiled coil structure. Each of them was acidic, trans membrane, hydrophilic and non-secreted protein. The secondary structure and three-dimensional structure which were mainly surrounded with a-helix and irregular curling of these two proteins were similar, but there were some differences on phosphorylation sites.3.2Cloning of Mn-SOD genes10different mRNA transcripts of Mn-SOD gene which were cloned from’Premier’Honey Peach EC were obtained in the experiment. The full-length sequences of cDNA were from960bp to1200bp. In order of G+C content, it was3’UTR>ORF>5’UTR. The sequence feature analysis showed that the10different mRNA transcripts existed4different3’untranslated regions and3different5’untranslated regions. The result showed that the3’untranslated regions and5’ untranslated regions of the sequences of Mn-SOD genes from’Premier’ Honey Peach EC had the characteristics of polymorphism。10different mRNA transcripts of the Mn-SOD gene encoded only1protein. The protein was consisted of260amino acids. The bioinformatics analysis revealed that might not have coiled coil structure. It was a alkali, transmembrane, hydrophilic and non-secreted protein. Secondary structure of protein was mainly composed of48.90%%α-helices,10.34%chain extension and38.46%irregular curling. Three-dimensional structure was mainly surrounded with a-helix and irregular curling. 3.3Cloning of Cu/Zn-SOD genes4different mRNA transcripts of the Cu/Zn-SOD gene family which were cloned from ’Premier’ Honey Peach EC were obtained in the experiment. The full-length sequences of cDNA were from860bp to900bp. In order of G+C content, it was3’UTR>ORF>5’UTR. The sequence feature analysis showed that Cu/Zn-SOD gene had3different3’untranslated regions.These mRNA transcripts of the Cu/Zn-SOD gene encoded only1protein. The protein was consisted of223amino acids. The bioinformatics analysis revealed that might not have coiled coil structure. It was a acidic, transmembrane, hydrophobicity and secreted protein. Secondary structure of protein was mainly composed of9.42α-helices,35.84%chain extension and54.71%irregular curling. Three-dimensional structure was mainly surrounded with β-fold and irregular curling.