The Overexpression Of Mz₂NHX₁ Gene In Malus Zumi

Malus zumi [(Matsum)Reder] is the hybrid cultivar of Malus manchurian and Malus sieboldii.Malus zumi is a kind of excellent apple rootstock which has stronger stress and saline-alkali resistance.Many cultivation tests on saline-alkali soil proves that Malus zumi has higher ability of salt-tolerance than some other salt-tolerant plants as well as higher disease resistance and graft compatibility.Meng xiaoye separated the Na+/H+ antiporter gene from Malus zumi in 2009,registeredand analyzed its expression feature(Gene Bank accession number: GQ 503257).On the basis,our laboratory separated its homologous gene:Mz2NHX1,sequenced and registered as well as constructed the expression vector:p RI201-AN-GUS DNA-Mz2NHX1.The study firstly established the system of ultrasonic-mediated transformation as well as the regeneration system by callus differentiation,by which the Mz2NHX1 gene was over-e xpressed.Results of GUS,RT-PCR testing and q-PCR testified that Mz2NHX1 gene was overexpressed.17 transformants were obtained and their physiological indexes like the activity of antioxidase was preliminarily analysed which was proved to be raised.It showed that the Superior medium for callus induction is: MS+6-BA 3.0mg/L+NAA 0.1mg/L and the optimal medium for callus differentiation and regeneration to a new plant is: MS+6-BA 2.0mg/L+NAA0.1mg/L.And the most efficient ultrasonic-mediat-ed condition is that treatment of 6s,intermittent of 10 s for 20 times by power of 80 w in which efficiency of transformation was up to31.3%.Transformants were obtained through the callus differentiation and regeneration system of Malus Zumi established before.The results of the detection of tranformants of Malus Zumi of GUS staining and RT-PCR both are positive.1635 bp band was amplificated and Mz2NHX1 gene was over-expressed distinctly over the control after RT-PCR appraisal.The quantity of expression of Mz2NHX1 gene was analyzed by q PCR.Results showed that the quantity of expression of Mz2NHX1 gene was 6.21 folds over the control.Transformants after appraised and the control were cultivated on medium containing200 mmo L/L of Nac L for 3 weeks,Results showed that transformants were better in growth with bigger leaves and so on,and leaves of the control showed withed and yellow leaves.The results of SOD showed that its activity in transformants was about 2 folds over the control,which suggested better system of clear of activity oxygen.And the activity of POD was 1.3 folds over the control,which indicated that transformants were more salt-tolerant.The results of MDA content in transformants and the control showed that the control was 1.5folds over the transformants.MDA is one of the products of membrane lipid.The resuls showed that it is less in the transformants,which means that transformants were less harmed by salt.The results of this research offer certain basis foundation for the improvement of physicochemical property and the utilization of saline-alkali soil,as well as for the breed of new salt-tolerant species of Malus Zumi.