| Japanese apricot(Prunus mume Sieb.et Zucc.),belonging to Prunus L.in Rosaceae,has been widely cultivated in Asia for approx.3,000 years(Chu,1999).It possesses a relatively large range of chilling requirement from 26.3CP to 75.7CP,making it an appropriate target for research into the mechanisms of woody plant dormancy.Many studies suggested that GA4 plays a key role in the regulation of dormancy release.In this study,we proposed that H2O2 act as a signal molecule in the GA4 treated dormancy realease.When Janpanese apricot was used as plant material,and using UV-photometric method and qRT-PCR technologies,we investigated the H2O2 concentration,variations in the activity of antioxidant enzymes,and variations of expression level in antioxidant enzymes encoding genes and signal-related genes up-or downstream.Effect of H2O2 on dormancy release in Japanese apricot buds induced by exogenous GA4 was analyzed.When tissue culture seedlings of polar was used as plant material,PmRGL2 overexpression vector was constructed and transformed into poplar using agrobacterium-mediated method.Using leaves of the transgenic polar as material,H2O2 concentration,activities of antioxidant enzymes were investigated.Expression analysis of genes which encoding antioxidant enzymes and signal-related genes were also performed.Using method of high performance liquid chromatography-tandem mass spectrometric(HPLC-MS/MS),content of GA4 in leves of transgenic polar and wild type polar were also investigate.The main results are as follows:1.High chilling requirement Japanese apricot cultivar of ’Bungo’ was used as plant material,and branches were collected for many times in the years of 2013 and 2014,during endodormancy.And then branches were treated by 10mM H2O2,200 μM GA4 and 1%H2O2 in a phytotron with conditions fit for growth.Changes of bud germination rates,H2O2 content,activities of antioxidant enzymes and the expression of corresponding genes and related genes of signal transduction were analysised.The results showed that germination rate was significantly higher in flower buds treated by 200 μM GA4 than flower buds treated by 1%H2O2,10mM H2O2 and distilled water,and the best time for 200μM GA4 treatment was around the New Year’s Day in Nanjing.Content of H2O2 and activity of SOD reached to a peak at the time of bud dormancy release,and expression levels of Respiratory intracellular hydrogen peroxide produced burst oxidase homolog protein E(NADPHoxi-E)and superoxide dismutase[Cu-Zn](SOD[Cu-Zn])were the highest at the same time.Catalase(CAT)and peroxidase(POD)has a role of scavenging H2O2.Activities of both the two enzymes are suppressed,besides activity of CAT was suppressed before dormancy release and activity of POD was suppressed at the time of dormancy release.But expression levels of genes(Probable phospholipid hydroperoxide glutathione the expression of Peroxidase and Catlase1-like)encoding the two enzymes were maintained at a high levels around the stage of dormancy release.Other genes expressed in flower buds of Japanese apricot after 200μM GA4 treatment were also significantly different from the other treatments and control,these including:GAI which encoding DELLA protein,GID1B-like which encoding GA receptor protein,Ca/calmodulin-dependent protein kinases related kinase 5(CDPK-5),Mitogen-activated protein kinase 9(MAPK-9)and Calreticulin-3-like(CRT-3-like).Based on the awareness that H2O2 may act as a signal molecular in dormancy release,we proposed that GA4 may regulate changes of H2O2 content through the variation of genes encoding antioxidation enzymes,and lead to expression level changes of signal related genes up-or downstream.And finally contribute to dormancy release.2.Two years old grafted seedlings of high chilling requirement Japanese apricot cultivar of ’Bungo’ and low chilling requirement Japanese apricot cultivar of ’Taoxingmei’were used as plant material.Chilling requirement was recorded using the 0～7.2℃ model.Seedlings were treated by GA4 in controllecd environment chamber for many times untill chilling requirements were satisfied.Germination rates of vegetative buds in the two cultivars were tested.And H2O2 content and activity of related antioxidant enzymes in vegetative buds were also been invesgated.The results showed that germination rate of vegetative buds in GA4 treated buds were significantly high than in control.And the best time for 250 μM GA4 to treat ’Taoxingmei’ was at one third of its chilling requirement.And the best time for 300 μM GA4 to treat ’Bungo’ was at the chilling hour accumulation has reached about half of its chilling requirement.In this study,we observed that the activity of antioxidant enzymes within two cultivars both rised after 15d treatment of GA4 and control,but antioxidant enzyme activity in vegetative buds was significantly higher in GA4 treatment than in control.Activities of CAT and POD in low chilling requirement Japanese apicot cultivar of ’Taoxingmei’ has higher level than in high chilling requirement Japanese apicot cultivar of ’Bungo’,and SOD activity has no significant difference between the twp chultivars.This suggested that both CAT and POD play an important role in dormancy release of vegetative buds.According to these results,we proposed that dormancy release in vegetative buds has similar mechanism with in flower buds,and both may regulate content of H2O2 through the regulation to antioxidant enzymes activity.3.PmRGL2 overexpression vector was constructed and transformed into poplar using agrobacterium-mediated method.Vector of pYH4215 used in this experiment has hygromycin resistance,so the transgenic seedlings can survive on a medium containing hygromycin.To further determine the hygromycin resistance poplar plants are indeed transgenic plants,method of GUS staining was used.We also extracted genomic DNA of transgenic poplar leaves,and using specific primers designed according to nucleotide sequence of hygromycin gene in pYH4215 vector for PCR detection.While exogenousPmRGL2 in transgenic poplar was also tested by PCR,preliminary results show that the gene has been successfully transferred PmRGL2 into poplar genome.Leaves of transgenic poplar seedlings and wild type seedlings were used as plant materials,and GA4 content,H2O2 content,activities of antioxidant enzymes were investigated,expression analysis of genes which encoding antioxidant enzymes and genes related to GA signaling transduction were also performed.We observed that expression level of the NADPHoxi gene,which encoding NADPH oxidase,in transgenic seedlings were significantly lower than in wild type seedlings,also were the GA4 content and H2O2 content;expression level of antioxidant enzymes encoding genes and activities of these enzymes has relatively high level in transgenic seedlings than in wild type seedlings;expression levels of GA20oxi and GA3oxi,which related to GA synthesis were significantly lower than in wild type polar;while,expression levels of GID1 gene,which encoding Gibberellin receptor protein,was sifnificantly higher than in wild type polar;expression level of genes including Mitogen-activated protein kinase(MAPK)gene and CDPK-related kinase 1 family protein(CDPK)gene,which related to signal transduction,were significantly lower in transgenic seedlings than in wild type seedlings;expression level of Calreticulin family protein(CRT)gene was significantly higher in transgenic seedlings than in wild type seedlings.These result suggested that over-expression of PmRGL2 genes in transgenic seedlings can inhibit the expression of NADPHoxi and then reduce the production of H2O2,and it may also improve the activity of antioxidant enzymes and lift the expression level of genes encoding antioxidant enzyme,thus will help to maintain the content of H2O2 in lower level.Then,lower level of H2O2 will have an effect on the variations of GA4 content and the expression levels of genes related to GA synthesis and signaling transduction.And ultimately,these will have an important effect on the development of transgenic polar. |
PDF Full Text Request