Study On Transformation Of The Gene AmEBPl From Ammopiptanthus Mongolicus Into Apricot

There are many resources about apricot seeds which are widely distributed in China.According to the cold spring and late frost in the North，a seriously impact on the flower andyoung fruit，which has a great damage to the products and economic benefits. So it is verygreat important to the cold-resistance breeding on apricot. Traditional plant breedingapproaches have had limited success in improving the cold tolerance. It cost a long time tobreed cold-resistance apricot with traditional method，even had no obvious effect. Much moreresearch focus on the cold resistant physiology about the apricot. Using the modernbiological technology to improve the cold-tolerance of plants with higher efficency andspecility, can accelerate the breeding process.The cold-resistance about the plant, is quite closely related to the synthesis of proteins underlow temperature conditions.The gene encoding ErbB3binding protein of Ammopiptanthusmongolicus（AmEBP1）associated with cold stress, which could inhibit the plant translationfactor（eIF2a）out of phosphorylation. AmEBP1also regulated the activity of the eIF2a tomake sure protein synthesis, which improved the cold resistance ability of the plant. In thisresearch, we designed specific primers by the nucleotide acid sequence of Ammopiptanthusmongolicus in Genbank. The gene coding sequence of AmEBP1were cloned from the totalRNA of the Ammopiptanthus mongolicus, then put the AmEBP1transformed into Daguoapricot to cultivate transgenic plants by immature embryos tissue culture. The main studywere as follows:1Ammopiptanthus total RNA was extracted from its leaves and the cDNA ofAmmopiptanthus gene AmEBP1was amplified from its total RNA by RT-PCR method.2The prokaryotic expression vector pET-22b-AmEBP1was constructed,which had beentransformed into E. coli. The survival rate of transgenic E. coli was higher than that of thenontransformed of E. coli at low temperatures.3Daguo,Daiyu and the other6apicot plants had significant difference on incomplete flowerratio. Daguo had the lowest rate at11.62%, but Honghebao highest above80.91%, Daiyu50.08%，Jintaiyang27.22%，Xinshiji25.43%. Daguo had the highest rate of set rate of fruit about29.34％, the second higher was Jintaiyang, the lowest was Honghebao, and7.23%onDaiyu.4A tissue culture system about apricot was established taking the young Daguo embryo formaterial.WPM medium was choosed as the basal culture medium for the young embryo ofDaguo with sucrose10g L-1，agar8g L-1，and pH5.8. WPM+6-BA0.8mg L-1+NAA0.2mg L-1was the differentiation medium to produce sprout and induce callus for buds;the best rooting medium was WPM+NAA0.1mg L-1+6-BA0.8mg L-1+IBA0.2mg L-1;5The plant expression vectors pCAMBIA2300-AmEBP1was constructed,which wassucceed to transform into Daguo embryos by pollen tube pathway and embryonic callus byparticle gun.Then the transformed material firstly grew in the WPM+Kan50mg L-1to getresistant plants,which secondly grew in the differentiation medium WPM0.8mg L-1+NAA0.2mg L-1to induce buds group to PCR. The last ones were put ionto the rooting medium togrew transgenic lines.6In total,6PCR testing plants were obtained from the kanamycin resistant ones.The put the6plants identified by Southern hybridization，then6lines showed positive hybridization results.These results indicated that the AmEBP1gene has integrated into the genomic DNA oftransgenic Daguo. RT-PCR analysis on line1and6which had single copy which showed thatthe imported AmEBP1gene expressed at transcriptional level in transgenic plants.7Put the transgene-positive lines1and6induced in the differentiation medium to inducebuds, then PCR identified the group buds, removed the negative ones, continued to inducedifferentiation. When got a lage number of materials,grew in the certain base to rooting fornext cold test. Results of the cold resistance tests pointed out that in the low temperaturetreatments（-4and-8℃）, the transgenic plants exhibited higher survival rates than theuntransformed control plants．Under the lower temperature cold treatment，the REC and MDAof transgenic plants was obviously lower than that of untransformed control plants. But thesoluble protein of transgenic lines was greatly higher than that of untransformed control lines.The LT50of transgenic lines was remarkable lower than that of the untransformed controlplants by2.13℃. The soluble protein, soluble sugars, free proline and MDA determinationresults of transgenic plants also supported that the imported AmEBP1gene increased the coldresistance of the Daguo apricot．8There are no target genes founded in the DNA of the rhizosphere soil and rhizosphere plantsabout the transgenic lines; Initial test of rhizosphere soil microbial effects of transgenic plantshowed that transgenic and non-transgenic lines modified strains of microorganism are bacteria, fungi and actinomycetes. There are no significant differences in the number, neitherin the varieties of the tested soils.