Studied On DIG-labeled Southern Blot Analysis And Cold Resistance Correlation Characteristics In Transgenic Lines Of Apple Carrying Exogenous CBF Gene

In this study, the transgenic plants of cultivar Gala carrying exogenous CRT/DRE binding factor gene were examined by PCR, PCR-Sourthern and RT-PCR.After cold acclimation, freezing tolerance was preliminarily evaluated by anatomy structure, relative electrical conductivity of leaves in vitro, proline contents, malondialdehyde contents, the activity of SOD and POD. The main results were as follows:1 The transgenic plants of cultivar Gala carrying exogenous CRT/DRE binding factor gene were tested by PCR analysis and showed specific positive band as same as the plasmid control, which proved that exogenous CBF genes were successfully transformed into apple.2 PCR-Southern with digoingenin labeled DNA probe results showed that exogenous CBF gene were successfully transformed into apple.3 RT-PCR analysis indicated that the expression of exogenous CBF gene was shown only in the transduced cells.4 Cross sections were prepared from leaves of transgenic plants and negative control grown at 25℃and after 2 weeks of cold acclimation at 2℃. The anatomy structure of leaves showed that palisade tissue against spongy tissue and CTR of the transgenic lines was the highest at 2℃, and SR was the lowest; palisade tissue against spongy tissue and CTR of the negative control was the lowest at 25℃, and SR was the highest; there were two layers palisade tissue in the transgenic plants cells after being 2℃, and there was one layer palisade tissue of the others.5 After cold acclimation, the relavity electrical conductivity of the transgenic plants was significantly lower than the negative control when after being in-8~-2℃, and as the treating temperature decreased, the relavity electrical conductivity was increased. LT50 of the transgenic plants was an average of -6.20℃, and LT50 of the negative control was -1.00℃.6 After cold acclimation, the activity of SOD of the transgenic plants was higher than unacclimated while he activity of SOD of negative control was lower than unacclimated. During -8~0℃, the activity of SOD and POD of the transgenic plants was significantly higher than of the negative control.7 After cold acclimation (0,-2,-4,-6,-8,-10℃), The content of MDA of the transgenic plants and negative control was increased as the temperature decreased, and all reached the peak at -6℃. The content of MDA of the transgenic plants was significantly lower than of the negative control under -6~0℃. The accumulation amount of MDA of the transgenic plants was lower than of the negative control.8 After cold acclimation (0,-2,-4,-6,-8,-10℃), the proline contents of proline of the transgenic plants was significantly higher than of the negative control under -6~-2℃. The proline contents of the transgenic plants and negative control was increased at first, and the accumulation amount of proline of the transgenic plants was significantly higher than of the negative control.9 When the temperature was decreased in greenhouse, the transgenic plants were defoliated while the negative control was not defoliated.