Characterization Of MdCSP3 In Response To Different Stresses

Apple(Malus × domestica)is one of the most important fruits worldwide.Northwest region of China is the largest and best region for apple production in China.However,annual precipitation is less and distributed unevenly in this region.In addition,extremely low temperature,deleterious diseases and deprived soil nutrient also seriously restrict the apple quality and production in this region.Therefore,identification of stress responsive genes in apple and explore their functions can provide genetic and theoretical basis for breeding of apple with stress resistance.In this thesis,we identified and cloned MdCSP3(COLD SHOCK DOMAIN PROTEIN 3)gene in ‘Golden Delicious'(Malus × domestica Borkh.cv.Golden Delicious)and then obtained two overexpression transgenic apple lines.We observed the phonotypes of the transgenic plants under drought,cold,nutritional deficiency and fungal disease.The results are as follows.1.In this paper,we cloned CSP3 in apple,which encodes 159 amino acids.Protein sequence analysis showed that MdCSP3 encoded an RNA binding protein with one RNA binding domain.By analyzing the expression level of MdCSP3,we found that MdCSP3 was expressed in root,stem,leaf,flower and fruit,with the highest expression level in root.The expression level of MdCSP3 was gradually increased under cold,heat and ABA stress,whereas decreased under drought and NaCl stresses,indicating that MdCSP3 might play roles in the stress response.2.MdCSP3 overexpression transgenic apples were sensitive to drought.Under drought treatment,MdCSP3 overexpressed transgenic lines lost water faster.In MdCSP3 overexpression transgenic lines,the expression level of MdDREB2 A ? MdNAC1,MdNCED3 and MdSnRK2.4,key gene in the ABA synthesis and transduction pathway,was significantly lower than that of non-transgenic plant GL-3 under drought stress.Therefore,MdCSP3 may negatively regulate apple drought resistance by negatively regulating ABA content and drought positive regulators such as MdDREB2 A and MdSnRK2.4.3.Compared with GL-3,MdCSP3 overexpression transgenic lines were more resistant to low temperature.Under low temperature stress,the expression level of the positive regulatory gene related to cold stress such as MdWARK6 in the CBF independent pathway was significantly higher in MdCSP3 overexpression transgenic lines than that in GL-3.Onthe contrary,the expression level of the negative regulatory gene related to cold stress including MdSOC1 ? MdJMT and MdESM1 in the CBF independent pathway was significantly lower in MdCSP3 overexpression transgenic lines than that in GL-3,indicating that MdCSP3 regulated freezing tolerance of apple by modulating expression of cold-responsive genes in the CBF independent pathway.4.In the nutritional deficiency experiment,it was found that the MdCSP3 overexpression transgenic apples were more sensitive to nitrogen starvation,and the expression level of nitrate reductase MdNR in the MdCSP3 overexpression transgenic apples was significantly lower than that of the wild type.Therefore,MdCSP3 may reduce the nitrogen absorption capacity of plants through NR.5.After inoculation,it was found that MdCSP3 overexpression transgenic apples were more sensitive to Apple Alternaria Blotch,indicating that MdCSP3 negatively regulates apple resistance to Alternaria Blotch.