| Peach is the fruit tree of the Rosaceae Prunus genus,which is one of the main planted fruit trees in northern China,and a considerable number of varieties have the problem that they are easy to be affected by low temperature,then the peach would be reduced and the tree would be death.In the production,the strong cold resistant peach varieties are urgently needed in the northern China,so improving the cold resistance of peach tree is one of the main objectives of peach breeding.In this experiment,the leaves and the flower bud,phloem were used as experimental materials,such as the strong cold resistance varieties,‘Maotao',‘Hunchun',‘Xin21shiji' and the week cold resistance varieties,‘21shiji',‘Zhonghua shoutao',‘You25-3'.PpFe-SOD genes in different cold resistant varieties were analyzed by gene cloning and bioinformatics,and the relative expression of PpFe-SOD gene in the flower buds and phloem at different stages from different varieties were analyzed by qRT-PCR,in order to understand the relationship between the SOD enzyme activity from the different cold resistant peach tree,the SOD enzyme activity was measured,then the pBI121-Fe-SOD gene expression vector was constructed and converted it to the tobacco by the agrobacterium-mediated method.The research results were as follows:1.The comparison of the gene and amino acid sequences of six peach varieties showed that there were single base differences among ‘Maotao',‘21shiji' and‘Zhonghuashoutao' compared with other varieties,but the conserved domains of six amino acid sequences were identical(18-319 bits),including a complete open reading frame with a total length of 960 bp,encoding 319 amino acids.Genetic evolution analysis showed that the PpFe-SOD amino acid sequence was similar to the rosaceae plants,such as prunus dulcis,prunus avium,prunus mume,malus domestica,rosa chinensis,the homology was more than 85%.2.The relative expression of PpFe-SOD gene increased in the beginning and then decreased in flower buds of different varieties,the trend was same as the trend of temperature,The results showed that PpFe-SOD gene was not directly involvedthe cold resistance regulation of peach blossom bud.The relative expression of PpFe-SOD gene in phloem of different varieties in different sampling periods showed two trends,‘Hunchun' and ‘21shiji' showed a gradually increasing trend,‘Maotao',‘Xin21shiji',‘Zhonghuashoutao',‘You25-3' showed a low-high-low trend,it suggested that the expression of PpFe-SOD gene in phloem may be related to cold resistance.The results showed that the mechanism of PpFe-SOD gene in flower bud and phloem was different.3.The flower bud SOD enzyme activity in different periods of six varieties showed the “high-low-high” trend,in contrast to the trend of temperature.The phloem SOD enzyme activity showed two trends,The SOD enzyme activity of‘Hunchun' and ‘21shiji' were increased gradually and the SOD enzyme activity of‘Maotao',‘Xin21shiji',‘Zhonghuashoutao',‘You25-3' were increased at first then redused.So it was speculated that SOD activity might be related to the cold resistance of peach.4.The transgenic vector pBI121-Fe-SOD was constructed,and successfully obtained the transformation of tobacco positive plants by agrobacterium-mediated transformation.At 5? low temperature,the growth of transgenic positive tobacco plant 7,10 and 12 was stronger than the wild type,the expression of PpFe-SOD gene in transgenic positive tobacco was higher than that in wild type,the change trend was high-low-high-low,the results showed that transgenic tobacco improved resistance to low temperatures,and the PpFe-SOD gene was speculated to be correlated with cold resistance of peach. |
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