Study On Transformation Of Galactinol Synthase Gene From Ammopiptanthus Mongolicus (AmGS) Into Tea-like Tree Photinia×Fraseri 'Red Robin'

Cold stress is a major environmental factor that limits the agricultural productivity of plants.Low temperature has strong impact on the survival and geographical distribution of plants.Cold stress often affects plant growth and productivity, which causes significant losses.Traditional plant breeding approaches have had limited success in improving freezingtolerance.Using the modern biological technology to improve the cold-tolerance of plants(especially woody plants) with higher efficency and specility, can accelerate the breedingprocess.An antifreeze gene AmGS (galactinol synthase gene of Ammopiptanthus mongolicus) wasisolated from Ammopiptanthus mongolicus, which has strong cold-resistance and couldendure low temperature below-30℃. In this study we obtained whole cDNA sequence thencloned into pET-22b (an prokaryotic expression vector) and pCAMBIA2300-35S-OCS (aneukaryoticexpression vector) respectively. A bacteria transformation vector pET-22b-AmGSand a plant transformation vector pCAMBIA2300-AmGS were constructed respectively. Andthen were transformed into E.coli and Photinia×fraseri, obtained the transgenic E.coli andtransgenic Photinia×fraseri of AmGS gene.The major results are as follows:1. The AmGS gene was successfully transformed into E. coli strain. The survival rate oftransgenic E. coli was higher than that of the nontransformed of E. coli at low temperatures.This fact support that the cloned AmGS gene is correct.2. Established efficient regeneration system of Photinia×fraseri. The best explant was waterculture shoots. Best initial medium was MS+6-BA1.0mg/L+NAA0.1mg/L, Formulacombination of adventitious bud induction was MS+6-BA2.0mg/L+NAA0.5mg/L, thisformula had a large number of axillary buds, sturdy seedlings, the color dark green.The bestmedium for regeneration differentiation of stem segments was MS+6-BA2.0mg/L+NAA0.1mg/L.IBA concentration as the main rooting culture limiting factor, the best rootingmedium was MS+NAA0.05mg/L+IBA0.1mg/L+sucrose10g/L.3. The plant expression vectors pCAMBIA2033-AmGS was constructed.We investigated themortality rate,differentiation rate,differentiation number of buds,sprouts length,Shoot color of transgenic plants.Found that impact site was the the major factor which effect oftransformation.The best Agrobacterium-mediated AmGS gene transformation program wasthat,Select shoot tip, with Agrobacterium stock solution (OD600≈0.4)1/2concentration, dip10min, put on the MS differentiation medium additional25mg/L acetosyringone (AS), at25℃under dark conditions,2d, then transferred to the concentration of50mg/L kanamycinMS medium filter culture.4. In total,32kanamycin resistant plants were obtained. From them, seven lines (R6, R7, R8,R9, R10, R11, R12) were identified by PCR, four (R6, R7, R8, R10) showed PCR positive,which were futher identified by Southern hybridization. Results indicated that3lines (R6, R7and8) showed positive hybridization results. These results indicated that the AmGS gene hasintegrated into the genomic DNA of transgenic plants of R6, R7and R8lines. RT-PCRanalysis on R6and R7showed that, the imported AmGS gene expressed at transcriptionallevel in transgenic plants.5. Transgenic plant heredity and genetic stability examination found that, in C1, C2and C3subculture plants, few plants appeared in PCR negative results, from C4to C6subcultureplant could not appear PCR negative plants. These results indicate that the transgene AmGScan be transferred to the offspring in the transgenic lines R6, R76. Cold resistance phenotype analysis showed that, under different temperature conditions,transgenic plants showed higher survival rate and much better cold resistance ability thancontrol wild-type plants.It proved AmGS gene enhanced the cold tolerance of transgenic plant.7. REC(relative electric conductivity)test results showed that, after low temperaturetreatment, the REC increasing level of transgenic plants was obviously lower than that ofuntransformed control plants. The LT50of R6strain was-12.93℃, R7strain was-12.63℃,untransformed control plant was-8.25℃. The LT50value of transgenic strains was obviouslylower than that of the untransformed control plants. These results proved that transgenicstrains cold resistant ability has been improved.The soluble protein, soluble sugars, free proline and MDA determination results of transgenicPhotinia×fraseri also support the harm suffered reduce after cold treatment, reflected that thecold resistance of transgenic plants improved.8. Initial test of rhizosphere soil microbial effects of transgenic plant showed that transgeniclines and genetically modified strains of microorganism are bacteria, fungi and actinomycetes,bacteria as the main microbial community. Transgenic plant rhizosphere soil microbialquantity change, in which the bacterial flora changes in maximum, but had little effect on the overall composition.Still bacteria has the largest number, fungi and Actinomyces in thesecond, Bacillus bacteria mainly in the bacterial flora.This study provide important theoretical reference and technical reserves to using geneticengineering techniques improved cold resistance of trees.This study screened R6, R7twotransgenic cold resistant lines and provides a selection of materials. Further research is still inprogress.