Effects Of Cold Stress On Physiological Metabolism And Isolating Cold-Regulated Genes From Euonymus Japonicus 'Zhuzi'

This paper focused on two aspects: the physiological effects of cold stress on the Euonymus japonicus 'Zhuzi'; the isolation of cold-induced cDNA gene. The results obtained were as follows:First, the chlorophyll (Chi) and carotenoid(Car) contents in Euonymus japonicus 'Zhuzi' seedlings decreased greatly as the cold stress proceeded, accompanied by the sharply increasing contents of MDA and PMP. It shows that Chi and Car degradation under cold stress was correlated with the damage of the plasma membrane.Second, the activities of protective enzymes i.e. superoxide dismutase(SOD), peroxidase(POD), and ascrobate peroxidase(APX) exhibited down-up-down trends when treated with 2@ and 8@, and these activities sustained at a relatively high level . This indicated Euonymus japonicus 'Zhuzi' had relatively high adaptability to cold stress. However, SOD, POD, and APX activities increased rapidly in 6h and then decreased sharply afterwards under freezing temperature treatment. They are severely inhibited by stress under-8@.Third, under the low temperature stress, the net photosynthetic rate and stomatal conductance (Gs) decreased but intercellular CO2 concentration increased greatly. It means the non-stomatal factors limited the net photosynthetic rate..Fourth, the Fv/Fo, Fv/Fm, Yield and photochemical quenching coefficient(qp) decreased whereas non- photochemical quenching coefficient(qN) increased under cold stress. Combined the above results, there existed significant correlation among chlorophyll a fluorescence parameters (Fv/Fo, Fv/Fm, Yield, qp, qN and MDA contents, plasma membrane permeability in Euonymus japonicus 'Zhuzi' seedlings. So it can be concluded that the inhibition degree of primary conversion of light energy was closely related to membrane lipid peroxidation in Euonymus japonicus 'Zhuzi' under cold stress.Furthermore, Suppression Subtractive Hybridization (SSH) was employed for the isolation of cDNA fragments for Euonymus japonicus ' Zhuzi' differentially expressed genes, and forward suppression subtractive cDNA Library of cold-regulated genes was constructed . The seedlings of Euonymus japonicus 'Zhuzi' treated with low temperature were as tester and untreated seedlings as driver. Subtractive cDNA Library was differentially screened through cDNA macroarray, six hundreds and four cDNA clones were identified as cold specifically induced or highly expressed. After sequencing of 84 cDNA clones and removing redundant cDNAs , we obtained 36 cold-regulated unique cDNA clones. 12 cDNA clones were expected to be novel genes, because no sequence homology with any known sequences was found in GenBank databases.The full length cDNA EJ175 was consisted of 603 bp to have an open reading frame from 57 to 515 including initiator codon and termination codon and to encode 152 amino acid polypeptide. Comparing with database in GenBank, the results showed EJ175 had 56% homology to mRNA for a known Pollen allergen-like protein in Arabidopsis thaliana. EJ175 was proved to encode gene of Pathogenesis-related protein Bet v I family by conserve domain analysis. AH above showed that the full length cDNA EJ175 encoded Pollen allergen-like protein and could be induced by abiotic stress factors.