Cloning And Expressing Of GalLDH Gene Under Temperature Stress In Potato Leaves

L-ascorbic acid plays a multifunctional role in both plants and animals,and comprises one of the most abundant metabolites in green tissues. It is a major antioxidant that serves many functions in plants and has many proposed roles in plant growth and metabolism..In addition, AsA functions as a co-factor for enzymes and ethylene-forming enzyme as well as for 2-oxoaciddependent dioxygenases required for the synthesis of abscisic acid and gibberellic acid. It also preserves the activity of a number of different enzymes by maintaining prosthetic group metal ions in the reduced state.In order to explore the relationship between GalLDH transcription, GalLDH activity and ascorbate content in potato A 1738 bp cDNA encoding L-galactono-1,4-lactone-dehydrogenase(EC1.3.2.3,GalLDH) fragment was isolated from Solanum.tuberosum'Luyin1'by the method of RT-PCR.The sequence was accepted and released byGenBank(Accession number EF125868). Research on expression of different organs and the relationship between GalLDH transcription, GalLDH activity and ascorbate content in potato leaves under temperature stress was based on the gene cloning.The following results have been obtained:1.Sequence analysis revealed high homology with those of GalLDH from tomato,tobacco and pepper.2.It showed that the level of GalLDH transcription was more abundant in young leaves and mature leaves than in old leaves and stems.3.Results showed that GalLDH expression was induced by light ,the level of GalLDH mRNA was lowest at 6:00 and it was highest at 18:00.But the GalLDH activity was highest at 12:00,so there were other elements effect GalLDH activity4.The AsA content of potato leaves increased at 0h-6h and decreased at 6h-12h during the day in high temperature stress,in the dark AsA content increased slowly.In the same time, the level of GalLDH mRNA and GalLDH activity had the same patterns.So the GalLDH expression was the main reason of AsA content increasement in high temperature stress.But the level of GalLDH mRNA and GalLDH activity.5. The AsA content of potato leaves increased at 0h-6h and decreased at 6h-12h during the day in low temperature stress,in the dark AsA content was steady,it decreased in the dark.In this treatment,the AsA content of po- tato leaves under low temperature stress was higher than that in normal temperature .But the level of GalLDH mRNA and GalLDH activity were lower than that in normal temperature.So the AsA content of potato leaves increased in low temperature stress was not caused by GalLDHexpression.6. In low temperature stress, the high AsA content of potato leaves had a realtion with low DHA content,it may be the result of AsA regeneration.7. The GalLDH cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form antisense constructs. The constructs were first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method and verified by PCR. It was indicated that the GalLDH gene had been recombined into tobacco genome and antisense transgenic tobacco plants were obtained.