The research on ultrasonic mediated genetic transformation of the Mz_2NHX_1 gene enhancing salt tolerance of Malus zumi

SUN Tingmei;WANG Luping;ZHANG Yongli;QIN Jiahui;YU Mengnan;LI Ai;PENG Lixin;Key Laboratory of Pomology,College of Horticulture and Landscape,Tianjin Agricultural University;Tianjin Hanbang Plant Protectant Limited Liability Company;

Malus zumi;;Genetic transformation system;;Ultrasonic;;Plantlet regeneration;;Salt-tolerant gene;;Overexpression

【Objective】In order to improve the salt-tolerance of Malus zumi and establish a more efficientgenetic transformation system for Malus zumi, the study established an efficient ultrasonic-mediated ge-netic transformation system for the Mz_2NHX_1 gene from Malus zumi by connecting the gene with thep RI201-AN-GUS DNA to build a plant expression vector. The Mz_2NHX_1 gene is a full-length Na+/H+anti-porter gene which can be found on the vacuolar of Malus zumi. The establishment of the ultrasonic- medi-ated efficient genetic transformation system of Malus zumi can be applied to the improvement of soil salini-zation and further analysis of the function of the Mz_2NHX_1 gene. The method of ultrasonic-mediated genet-ic transformation of Malus zumi is more efficient as well as less false-positive descendants than the meth-od of Agrobacterium-mediated genetic transformation and it represented the first time for the study of theultrasonic-mediated genetic transformation of Malus zumi.【Methods】Cut the main vein of the leaves of Malus zumi with 3-4 incisions perpendicularly under aseptic conditions. Inoculate the leaves on the MSmedium with different concentration ratios of 6-BA and NAA. Cultivate the leaves first in darkness for 2weeks at 24 ℃, then in 3 000 lx of light for 12 hours every day at 24 ℃ for 4 weeks to study the optimalmedium components of the callus induction and differentiation of Malus zumi. Cultivate leaves of Malus zumi on MS medium containing different concentrations of kanamycin: 0, 20, 30, 40, 50, 60, 70 mg·L~(-1)re-spectively to study the sensitivity of the callus of Malus zumi to kanamycin. Prepare the reaction buffer ofthe ultrasonic-mediated genetic transformation for 50 m L containing DMSO(1.1 mg·L~(-1)) for 5m L, recombi-nant DNA(Mz_2NHX_1-p RI201-AN-GUS DNA) for 200 ng, sodium chloride for 3 mg and sodium citratefor 2 mg. Leaves of Malus zumi with 3-4 incisions were immersed in the reaction buffer. The probe of theultrasonic was 5mm underneath the fluid level of the reaction buffer processing with a series of workingpowers of 60 W, 80 W, 120 W and ultrasonic processing times of 10 times, 20 times and 30 times and theultrasonic processing conditions for each time were processing for 6 s then intermittently for 10 s. Culti-vate the ultrasonic-processed leaves on the medium of MS+6-BA 2.0 mg·L~(-1)+NAA 0.1 mg·L~(-1)+Kanamy-cin 50 mg·L~(-1)after washing by sterile water for three times and cultivated first in darkness for 2 weeks at24 ℃ then in 3 000 lx of light for 12 hours every day at 24 ℃ for 4 weeks to study the optimal ultrasonic-processing conditions. Prepare the reaction buffer of GUS staining for 1 m L containing Na2 EDTA for 20 μL(0.5 mol·L~(-1)), Triton X-100 1 μL, phosphate buffer(1 mol·L~(-1), p H=7.0) of 100 μL, K3Fe(CN)6(0.1 mol·L~(-1))of 5 μL, K4Fe(CN)6·3H2O(0.1 mol·L~(-1))of 5 μL, X-Gulc(10 mg·m L~(-1)) of 200 μL then supplemented to 1m L by ultrapure water. Incubated them at 37 ℃ overnight then decolored by 95% ethanol for three times.Use the Easy Pure Plant Kit to extract the total RNA of the leaves of Malus zumi which were positive withGUS staining. Store the RNA at-80 ℃. Prepare the reaction buffer of reverse transcription to syntheticc DNA for 20 μL containing RNA of 200 ng, Anchored olig(d T)18of 1 μL, d NTP(each10 mmol·L~(-1)) of 1 μL,Easy Script Reverse Transcriptase of 1 μL, 5×Easy Script Reverse Transcriptase buffer of 4 μL, Ribonucle-ase Inhibitor(50 units·μL- 1) of 0.5 μL, supplemented to 20 m L by using RNase-free Water. The proce-dure of Reverse transcription was Incubating at 42 ℃ for 30 min then 85 ℃ for 10 s to deactivate the Ea-sy Script Reverse Transcriptase. RT-PCR was processed with the c DNA product as the template. Preparethe reaction buffer of RT-PCR for 25 μL containing c DNA of 1.5 μL,Easy-Taq enzyme of 0.2 μL, 10×Easy-Taq Buffer of 2 μL, d NTP(each 2.5 mmol·L~(-1)) of 2 μL, reverse and forward primer(20 μmol·L~(-1))0.8 μL, supplemented to 25 μL by DEPC Water. The procedure of RT-PCR was 95 ℃ of 5 min for one cy-cle then 94 ℃ of 1 min, 50 ℃ of 1 min and 72 ℃ of 1 min for 30 cycles, at last Incubated at 72 ℃ for 10 min. The results were analyzed using the BIO-RAD gel imaging system. The quantity of expression of Mz_2NHX_1 gene was analyzed by q PCR. Transformants were appraised and the control were cultivated on amedium containing 200 mmo L·L~(-1)of Nac L for 3 weeks to analyze the salt-tolerance of the transformants.【Results】The superior medium for callus differentiation and regeneration is MS+6-BA 2.0 mg·L~(-1)+NAA0.1 mg·L~(-1), which has the highest rate of differentiation in this study. The optimal kanamycin concentra-tion of resistance selection is 50 mg·L~(-1)and the most efficient ultrasonic-mediated condition in this studyis the treatment of 6 s, intermittent of 10 s for 20 times by the power of 80 W in which the efficiency of thetransformation was 31.3% which was two folds higher than using the method of Agrobacterium-mediatedgenetic transformation. Positive seedlings were obtained through callus differentiation and resistance se-lection. Tranformants of Malus zumi were dyed in Blue after GUS staining while non-transformants of Malus zumi were white after decoloring. 1 635 bp band was amplificated and the Mz_2NHX_1 gene was overex-pressed after the RT-PCR appraisal in the transformants. The results of q PCR showed that the quantity ofexpression of the Mz_2NHX_1 gene was 6.25 folds greater than the control. The results showed that transfor-mants were better in growth with bigger leaves and so on, and the leaves of the control were shown to bewithered and have yellow leaves. The results showed that the activity of POD in transformants was 2 foldsgreater than the control, which indicated that the transformants were more salt-tolerant. The results of theMDA content in the transformants and the control showed that the control was 2 folds greater than thetransformants, which means that the transformants were less harmed by salt【.Conclusion】This study estab-lished a ultrasonic-mediated transformation system of the Mz_2NHX_1 gene form Malus zumi. The method isof lower cost, more simple operation and further avoided the false-positive descendants while the methodof agrobacterium-mediated genetic transformation is easier infected with Agrobacterium and leads to morefalse-positive descendants. More salt-tolerant plants of Malus zumi were obtained through the method ofthe ultrasonic mediated efficient genetic transformation system of Malus zumi, which offers a foundationfor further research of salt-tolerant genes and breeds of more salt-tolerant materials.