| Sucrose non-fermentation-1-related protein kinase(SnRK1)is a typical serine/threonine protein kinase in plants and it has high homology to yeast SNF1 and mammalian AMPK in structure and function.SnRK1 is one of the important pivots for regulating carbon and nitrogen metabolism as well as plant growth and development.However there are few studies on SnRK1 in fruit trees,and the molecular mechanism of its regulation is still not clear.Our previous research found that overexpression of MhSnRK1 in tomato increased the fruit soluble sugar content and promoted the fruit ripening,but the molecular mechanism by which SnRK1 regulates fruit ripening was unclear.In order to further understand the regulation of SnRK1 in fruit development,we used the Prunus persica var 'Ruipan 17' as a test material to study the changes of SnRK1 kinase activity and the expression levels of each subunit coding gene during fruit development.To further explore the role of SnRK1 in sugar metabolism pathway,we applied different exogenous sugar treatment and adopted different fruit thinning treatment to study the changes of SnRK1 protein kinase activity and its response to different sugar signals.Since the peach transformation system has not been effectively established,we studied the mechanism of SnRK1 protein kinase to regulate fruit maturation and development by means of the model plants,using the tomato homozygous plants with overexpression of PpSnRK1? as the test material.The structure of SnRK1 protein kinase is a trimeric complex,which consists of catalyzed subunits ? and regulated subunits ? and ?.At present,there are few reports on the function of regulating subunit ?,therefore,we cloned the PpSnRK1?1 gene,and studied its function by heterologous expression in the model plant Arabidopsis.The specific research results are as follows:1.The “Ruipan 17” was used as the test material,and the activities of SnRK1 protein kinase at different stages of peach fruit development were measured and the expression of each subunit coding gene was analyzed.It was found that the activity of peach SnRK1 protein kinase was higher at the early stage of fruit development and during the two rapid growthperiods,while the activity was maintained at a low level at the hard-core stage,which was consistent with the gene expression level of its catalytic subunit ? at the transcriptional level.2.Through exogenous trehalose and sorbitol treatment of peach fruit,it was found that SnRK1 activity could respond to the induction of two exogenous sugar signals.After 100 mM trehalose treatment of peach fruits,the content of trehalose-6-phosphate(T6P)in the fruit was significantly increased,while the activity of SnRK1 protein kinase was significantly decreased;Meanwhile,the activity of sorbitol dehydrogenase(SDH)and sucrose synthase in the synthetic direction(SS-s)was also significantly decreased,while the activity of sucrose phosphate synthase(SPS)was significantly increased.The activity of peach SnRK1 kinase was significantly increased under the same condition of sorbitol treatment,and SDH activity was also significantly increased compared with the control.Moreover,the content of fructose and sucrose in peach fruits was significantly increased after sorbitol treatment.Under the treatment of two different sugars,the changes of SnRK1 protein kinase activity showed opposite trend,but the changes of SDH activity was consistent with SnRK1 kinase activity,suggesting that there might be interaction between them.3.The interaction between peach SnRK1? and SDH and SPS was verified by yeast twohybrid assay.At the same time,the purified proteins of PpSnRK1 and SDH were obtained through prokaryotic induction protein expression,and the phosphorylation between SnRK1 and SDH was preliminarily verified by the method of SnRK1 kinase activity determination.It suggests that SnRK1 may be involved in the regulation of sorbitol metabolic pathway by regulating the SDH activity at the protein level.4.The activities of SnRK1 kinase in PpSnRK1? transgenic tomato lines OE-1,OE-3 and OE-4 were significantly higher than that in the wild type at different stages of fruit development.The content of starch and soluble sugar in ripening fruits was increased significantly,while the content of soluble protein and titrable acid was decreased significantly.At the same time,it was found that the PpSnRK1? transgenic tomato fruits were mature about 10 days earlier than the wild type.The yeast two-hybrid experiment demonstrated the interaction between PpSnRK1 and the transcription factor SIRIN protein,and BiFC assay further verified that the interaction between the two proteins in the plant occurs in the nucleus.5.Fluorescence quantitative results showed that the expression of RIN in OE-1,OE-3and OE-4 transgenic tomato fruits was significantly higher than that of the wild type since the veraison stage,and the upregulated expression was most significantly higher than that of the wild type in the pink period.At the same time,the expressions of RIN-downstream targetgenes related to mature such as NOR,FUL1,ACS2,ACS4 and E8 were also up-regulated accordingly,the encoding genes of transcription factor NOR and FUL1 showed different expression patterns during fruit development,the NOR had the highest expression level in the veraison stage and the FUL1 had the highest expression level in the red ripening stage;The ACS2 had the highest expression in the red ripening stage,while ACS4 had the highest expression in veraison stage.At fruit different development stages,the expression of NOR,FUL1,ACS2,ACS4 and E8 in the PpSnRK1? transgenic tomato lines OE-1,OE-3 and OE-4were all significantly higher than that in WT,and the ethylene release of the mature fruits was also significantly higher than that of the wild type6.According to the published gene information on the peach genome website,the molecular weight and isoelectric point of the PpSnRK1?1 subunit were analyzed by online software.The results showed that the molecular weight of PpSnRK1?1 subunit was 46.8 kDa and the isoelectric point was 5.12,and its subcells is localized in the cytoplasm.At the same time,the promoter sequence of PpSnRK1?1 gene was analyzed by PlantCARE software to investigate the regulation of PpSnRK1 at the transcription level.The sequence of about 2000 bp before the initial codon of PpSnRK1?1 gene was selected and analyzed.The results showed that the PpSnRK1?1 could not only respond to the induction of abiotic stresses such as temperature stress,but also to the induction of hormones such as ABA and JA.7.The activity of SnRK1 kinase in PpSnRK1?1 transgenic Arabidopsis lines A62,A63 and A65 was significantly higher than that of WT,which increased by 12.9%,18.8% and10.4%,respectively.High temperature stress experiments showed that the chlorophyll content of PpSnRK1?1 over-expressing Arabidopsis lines was significantly higher than that of wildtype plants after treatment at 37 °C for 6 hours,and the leaf yellowing degree was significantly lighter than that of wild-type plants.The activities of SOD and POD in PpSnRK1?1 over-expression Arabidopsis strains A62,A63 and A65 were significantly higher than that of wild type,while the content of MDA was significantly lower than that of WT.It shown that overexpression of PpSnRK1?1 in Arabidopsis can significantly improve the plant tolerance to high temperature stress. |
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