Cloning Glycinin Gy3 Gene And Transforming It Into Bromus Inermis Mediated By Ion Beam

A pare of primers was designed and synthesized according to the glycinin Gy3 cDNA sequence reported in Genbank (M36686). A DNA fragment of about 1520bp was amplified by RT-PCR from the total RNA of the cotyledon of the soybean "Hei nong 37", and then it was cloned into pMD19-T vector. Sequencing results showed that the DNA fregment is composed of 1523bp nucleotide, with homology 99% to that reported in Genebank (M36686), which confirmed that the coloned cDNA is glycinin subunit gene Gy3. Then the Gy3 was cut out of pMD19-T vector and ligated with the plant expression vector pBI121. The constructed expression vector pBI121/Gy3 cntains a CaMV35 promtor, NOS terminator and NPTâ…¡gene,and was transformed into bromus inermis mediated by ion beam, for the purpose of improving the quality of pasture.For the mutagenic effects of ion beam on Bromus inermis seeds, it was found that plant survival rate could be affected by different ions'energy. So we studied on the relationship between survival rate of seeds and energy dose, and found that the suitable transformation doses should be 800, 900, 1000×2.6×10¹³Ar⁺/cm².We transformed GUS gene mediated by ion beam into Bromus inermis seeds, but did not see its transient expression. Thus result disagreed with other's observation in which arabidopsis seeds were implanted with ion beam and transformed at 24～48h after germination, 10% transient expression were obtained. So we bourgeoned Bromus inermis seeds, and determined the suitable transformation doses as 300, 400, 500×2.6×10¹³Ar⁺/cm², however we didn't get the perfect result.We also studied how to avoid frostbite to the callus of Bromus inermis during ion beam implantation, and found the suitable transformation doses are 2.6, 5.2, 7.8×10¹⁵Ar⁺/cm², of which the best dose is 5.2×10¹⁵Ar⁺/cm². Finally we got the transformants.