Functional Analysis Of GPX、ZjPIP2and ZjMT Gene Of Ziziphus Jujuba

Jujube (Ziziphus jujuba Mill) is a unique fruit trees,with the3000years of cultural history.It is cold resistant, drought resistant,antioxidant,suitable to grow in poor soil.And these features of the jujube are significantly better than other crops. Therefore, the resistance gene of the jujube is the valuable genetic resources.This research is for the study of glutathione peroxidase genes,aquaporin gene,metallothionein genes and Metallothionein genes which is from branches cDNA library of Huping, tissue culture of zizyphus jujuba seedlings for the test material, analysis of3gene expression under abiotic stress, and constructs three genes of the plant expression vector, By transgenic methods,put the plant expression vector into the model plant of Arabidopsis,and get the cellular localization and functional identification of the gene. In order to reveal the resistant mechanism in the jujube,It also provides the theoretical basis and practical basis to make full use of the excellent genes to improve stress tolerance of plants.1、Jujube Glutathione peroxidase gene(1)Expression of the Glutathione peroxidase gene in Ziziphus jujuba Mill(2)The function analysis of Ziziphus jujuba Mill2、JujubeAquaporin genes (ZjPIP2)(1)Expression of the ZjPIP2in Ziziphus jujuba MillBy taking lajiao tissue culture rooting seedlings as test materials,select the same rooting of seedlings to simulated drought (PEG6000) and NaCl stress.And the fluorescent quantitative RT-PCR results showed that when the ZjPIP2in drought stress, expression levels increased at first and then decreased with increasing time and concentration.When in0.5MPa stress, in the first hour, the expression level rose in turn.After first hour expression began to decrease, and the48th hour to the minimum. When in0.8MPa stress,the expression of45th min rose to maximum,the followed reduced.When in1.2MPa stress, at45th min expression reached the largest amount,and then in turn reduced. When in the stress with the different PEG6000concentration,the ZjPIP2with lower concentration expression levels were maximum.By salt stress, expression with increasing time and concentration first increased and then decreased again increased.When in50mmol/L stress, in the first hour,the expression level rose to maximum.After the first hour expression began to decrease,but the24th and48th hour rose again.When in100mmol/L stress, in the first hour,the expression level rose to maximum.After the first hour expression began to decrease,but the48th hour rose again.When in200mmol/L stress, the45th min rose to maximum, and then decreased,but the24th and48th expression levels began to rise again. When in300mmol/L stress,expression levels reached maximum for the first time at45th min,and then began to decrease,but the expression level began to rise, and the expression reached maximum at24th and48th hour.In salt and drought stress,with increasing time and concentration,the(2)The function analysis of ZjPIP2We put the jujube ZjPIP2Aquaporin genes into plant expression vector PEZR(K)-LNY, built the YFP integration of express ZjPIP2and yellow fluorescent protein engineering strains of PEZR(k)-LNY-ZjPIP2-L.The19transgenic Arabidopsis plants containing the target gene ZjPIP2To generation were acquired.The target gene ZjPIP2transferred to Arabidopsis successfully was confirmed by PCR.T2generation of ZjPIP2transformation Arabidopsis seeds were sown in1/2MS medium with different concentrations of NaCl to evaluate their salt tolerance. It found that the ZjPIP2transformation Arabidopsis plants grown in salt stress and root were significantly better than the wild type (WT) Arabidopsis plants.The T2generation of ZjPIP2transformation Arabidopsis with two leaves and WT Arabidopsis plants were treated in salt and drought stress.After15days,to observe the growth conditions,it found that ZjPIP2transformation Arabidopsis was significantly better than WT plants in salt resistance and drought tolerance. The transgenic plants have sterile phenotype, Microscope observation of preliminary judging is caused by the Stamen development of transgenic plants.Cytological Observation to the transformation plants showed that there were lots of expressions of transformation Arabidopsis thaliana leaf epidermal cell membrane、cell nucleus and stomatal guard cell nucleus.3、Jujube Metallothionein gene(ZjMT)(1)Expression of the ZjMT in Ziziphus jujuba MillThen the RNA of each sample was extracted.And RNA analysis of fluorescence quantitative PCR after reverse transcription.By taking lajiao tissue culture rooting seedlings as test materials,select the well-growing crops and the same rooting of seedlings to simulated drought (PEG6000)、NaCl and CdCl2stress.Then the RNA of each sample was extracted.And RNA analysis of fluorescence quantitative PCR after reverse transcription.The results show that: after the ZjMT was coerced by PEG6000, the expression level increased with time and osmotic potential first increased and then reduce.When in0.5MPa stress with time increasing, the expression level rise in turn and45th min expression levels were maximum, then the expression levels began to decline,and48th hour expression levels were minimum. When in0.8MPa stress，45th min expression were maximum and then in turn reduced.When in the stress with the different PEG6000concentration,the ZjMT expression levels with lower concentration were maximum in each time.By salt stress, ZjMT expression levels with increasing time and concentration first increased and then decreased. When in50mmol/L stress,45th min expression levels were maximum. When in100mmol/L stress,45th min expression levels reached maximum.When in200mmol/L stress,the maximum expression levels were at30th min.When in300mmol/L stress,expression levels reached the maximum at30th min.By CdCl2stress, ZjMT expression levels with increasing time and concentration first increased and then decreased, and then increased and decreased again. When in100mmol/L stress, expression levels reached maximum for the first time at6th hour,and the second maximum was at24th hour,and the Minimum expression levels were at48thhour. throughout the process the expression of Transgenic jujube seedlings were higher than in the control group.Therefore, ZjMT is related to jujube’s drought resistant and salt tolerance and heavy metals resistant.(2)ZjMT’s salt tolerance identification and cellular localization in ArabidopsisWith the transformation of Arabidopsis by engineering bacteria PEZR (K)-LNY-ZjMT-L, we can get thaliana15To generation plants of ZjMT transformation Arabidopsis.It confirmed that the ZjMT was transferred into Arabidopsis successfully by PCR.T2generation of ZjMT transformation Arabidopsis seeds and wild type (WT) Arabidopsis seeds were sown in1/2MS medium with different concentrations of NaCl to evalue their salt tolerance.By observing the growth situation,it found that the ZjMT transformation Arabidopsis plants grown in salt stress and root were significantly better than the wild type (WT)Arabidopsis plants.By puting the transgenic Arabidopsis thaliana seeds in the heavy metal stress,it found that the seeds did not improve the heavy metal tolerance.The transgenic plants have sterile phenotype, Microscope observation of preliminary judging is caused by the Stamen development of transgenic plants.Cytological Observation to the transformation plants showed that there were lots of expressions of transformation Arabidopsis thaliana leaf epidermal cell membrane、Cell nucleus and stomatal guard cell nucleus.