The Production Of Vector-free And Marker-free Transgenic Soybean Expressing A Phytase Gene

Rapid development of plant transgenic techniques had impacted on traditional cropbreeding deeply. However, the field test of transgene crops has been slowered down in 1998with the issue of transgenics biosafty, particularly the concern for the unexpected biosaftyproblem from the vector such as its backbone sequence, resistant marker and promoter genesincorporated into it. The issue of transgenic plants biosafty attracts pulic concerns andrestricts the progress of transgenic crops industrialization. Therefore, developing vector- andmarker-free plant molecular breeding technics is one of the frontier areas in intrasngenicplants.Extracellular phytase produced by Aspergillus awamori 3.324 through semi-solidcultivation was purified by ultrafiltration followed by chromatography using ion exchange,gel filtration and chromatofocusing columns, sequencely. The purified enzyme is a 118kDaprotein including 40.6% glycosylation. It possesses optimum temperature and pH values of55℃, 2.5 and 5.5. The K_m and V_max of the enzyme for dodecasodium phytate at 37℃are 1.03nM and 2.13μM/min, respectively. Phytase activity was observed not affected by EDTA, andinhibited moderately by Ca²⁺, Mg²⁺, Mn²⁺, and significantly by Fe²⁺ and Zn²⁺. The enzymeexhibits better thermostability at high temperature than the commercial phytase product.The phytase gene was obtained by PCR amplification, and named as phyA (GenBankaccession no.DQ192035), with a size of 1515 bp and fungi characteristic intron sequence.phyA exhibits a high homology with filamentous fungi on both nucleotide and amino acidsequences. Compared with A.ficuum NRRL3135 and A.niger 963 phytase genes, itsnucleotide sequence shows 92.1% and 95.0% homologies (except its intron sequence, whichshows a big difference), while the amino acid sequence deduced from phyA shows 95.9% and94.0% homologies. The molecular weight of the phytase is 51042.8Da, which encodes apolypeptide of 467 amino acids. The amino acid sequences deduced from phyA contains theactive site septa-petide RHGXRXP, catalytically active dipeptide HD and 10 glycosylationsites, and 21 amino acids at N-ternimal are expected to be a signal peptides sequence.The ORF of the phytase gene was cloned into the plant expression vector pBI121. Therecombinat plasmid pBI121-phyA was then transferred into tobacco via the Agrobacteriummediation. The results of PCR and Southern Blotting showed that the phytase gene wasintegrated into the tobacco genome. The phytase activity assay of tobacco leavesdemonstrated that the recombinant phytase gene was highly expressed. The characterization of the recombinant enzyme expressed indicated the same temperature profile with the phytaseproduced by Aspergillus awamori, although slightly lower thermostability and one-unit shiftof the optimum pH were observed.A minimal linear transgenic DNA containing phyA gene without vector and marker wasobtained from the plant express vectorpBI121-phyA by PCR, which was introduced intosoybean by the pollentube pathway. A total of 279 flowers were treated with the lineartransgenic DNA. The PCR amplifications indicated that 13 in 99 T₁ plants contained the genecassette, with a transformation frequency of 13%, and 102 plants in 473 T₂ plants showedPCR-positive amplifications. T₃ individuals derived from the lines L14-11-2 and L14-19-1were investigated by Southern blot analysis, indicating their low copy number insertions.RT-PCR results showed the presence of phyA transcripts. The T₃ progenies from the linesL14-11-2 and L14-19-1 were collected to analyze phytase accumulation in their leaves. Thephytase level increased from week 2 to week 4, remained at stable for the following 2 weeks,but slightly decreased during week 7. Phytase activity in the plants from the line L14-19-1was higher than that from the line L14-11-2 from weeks 2 to 6. The highest expression wasobserved in the line L14-19-1 during week 4 (150 U/mg protein), about 3 times higher thanthat of the untransformed control (56 U/mg protein). In seeds, the transgenic seed displayed a100% increase in phytase activity compared to that of the wild-type. The temperature stabilityof the plant-synthesized recombinant phytase in the T₃ transgenic soybeans was analysised.Upon incubation at 60, 65 and 70℃for 10 min, 70%, 40% and 10% of the activity remained,respectively, for the crude extracts of the transgenic seeds, whereas no enzyme activity wasdetected for the untransformed control. Western-blotting showed that the recombinant phytasemigrated with an apparent molecular mass of approximately 73 kDa.In conclusion, the phytase gene was inherited and expressed in the recombvinant soybeansdeveloped with the vecter- and marker-free plant molecular breeding approach, through whichthe biosafty dispute could be overcome. The patent for this new technique was approved byState Intellectual Property Office of P.R.China (ZL 02 1 328382). Now the biosafty of gradeâ…¡was authorized to the two lines L14-11-2(1411) and L14-19-1(1412) by the Ministry ofAgriculture of P.R. China.