| For extending the processing period and reducing the loss of raw materials, low temperature was commonly used during the potato tubers storage. But the accumulation of reducing sugar (RS) in the tuber, called cold-induced sweetening (CIS), also will happen correspondingly with the temperature change and has become one of severe blockage of potato processing industry. Herein, two candidate genes, StGAPC and StEnolase, which were both up-regulated during the cold storage of CIS-resistant potato (S.berthaultii) tubers, were cloned. And the reverse genetics method was performed to verify the function of StGAPC and StEnolase on the metabolism of starch to sugar and CIS. Main results were as follows:1. The genes encoding cytoplasmic glyceraldehydes3-phosphate dehydrogenase (StGAPC) and Enolase (StEnolase) were cloned by RT-PCR from S. berthaultii. Their ORF was1017bp and1335bp that encoded a338-and a444-amino acid peptide, respectively. Both StGAPC and StEnolase showed constitutive expression pattern in different parts of potato and no organic specificity. However, both StGAPC and StEnolase were up-regulated in cold-stored tubers, indicating that they were involved in the response to low temperature of potato tuber. The subcellular localization revealed that StGAPC localized in the cytoplasm, while StEnolase was mainly in the membrane.2. The StGAPC transgenic lines exhibited no major phenotype changes in either plant morphology or tuber. But the tuber dormancy of RNAi lines were obviously shortened and the apical dominance of tuber buds during germination was disappeared and multiple buds of tuber sprouted at the same time in transgenic lines. The determination results of gene expression, enzyme activity and reducing sugar content showed that silencing of StGAPC could obviously decrease the GAPDH activity, and accelerate the respiration rate of cold-stored RNAi-tubers as well as the RS content. By contrast, over expression of StGAPC could reduce the RS content by nearly50%in some transgenic lines compared with the control. Furthermore, the results of metabolites measurement showed that inhibition of StGAPC expression led to the remarkable decrease of the downstream product of GAPDH catalysis (3-PGA) and the significant increase of some components of glycolysis and TCA. This suggested that StGAPC might influence the respiration rate and RS content of cold-stored tubers by changing of TCA and PPP pathway to regulate CIS a certain extent. However, further experiments are needed to confirm this hypothesis and explain the dormant shorten and the loss of apical dominance.3. No obvious variation of plant and tuber morphology can be observed between wild type and StEnolase over-expressed transgenic lines, but the RNAi-lines showed a lot of variation, such as foliage yellowing, growth inhibiting and tuber yield declining obviously, and the content of chlorophyll and RS as well as Suc were significantly decreased compared to the control. It indicated that suppression of StEnolase expression can influence the photosynthesis and finally affecte plant development. Further study suggested that both Enolase enzyme activity and PEP contents were reduced in RNAi lines, while glucose, fructose and sucrose concentration were significantly higher than the wild type stored in8℃for30days. However, the opposite results were not occurred in over-expressed lines maybe because overexpression of StEnolase was no high enough to modify the phonotype. The result of metabolite components showed the content of PEP and aromatic amino acid were both significantly lower than the control, but the content of malic acid, sucrose-6-P and trehalose-6-p were higher in RNAi lines compared to the control during the storage. All these indicated that StEnolase can also partly regulate the CIS and the aromatic amino acid synthesis pathway. Further confirmation is still needed to explain the reason of the increased respiratory intensity. |
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