Transferring Laccase Gene From Pycnoporus Sanguineus Into Tabacoo Mediated By Agrobacterium Tumefaciens

The genetic traits of transgenic plants improved technology,short culture period,income effect is good,can induce changes in plant traits,ornamental flowers and other fields has been widely used in crops.Laccase can be applied in papermaking industry,organic synthesis,food industry and other fields,small impact on the environment pollution and high efficiency.At present,the soil pollution control to a certain extent the use of microorganisms,but the engineering bacteria for transgenic strains,into a large number of possible biological pollution in the environment,an unforeseen losses,the use of phytoremediation method can avoid the occurrence of this situation.In this paper,in order to obtain the Pycnoporus sanguineus laccase gene as template,amplified the target gene fragment.The fragment was 1557 BP in length,encoding a protein of 519 amino acids,GC%58.15%,and GenBank in the Pycnoporus sanguineus laccase cDNA sequence comparison,the amplified sequence specific base mutation,but the corresponding amino acid did not change greatly change.The plant expression vector PBI121 with laccase gene to construct the recombinant expression vector PBI121-lac,in a large number of amplified in Escherichia coli DH5a expression,and transformed into Agrobacterium tumefaciens strain LBA4404.By using agro bacterium mediated transformation method of tobacco leaf tissue,to optimize the factors which effect the conversion efficiency.The results are as follows,in order to cultivate tobacco aseptic leaves growth of about 30 d as material,using MS as the basic medium,the different concentrations of hormone and select the best hormone ratio was 1 mg/L 6-BA and 0.1 mg/L NAA.The kanamycin resistance selection pressure for the screening of the organization,when the Kan concentration is larger than 100 mg/L,a large number of albino leaf differentiation rate of death,and will affect the plant tissue,a total of 80 mg/L Kan is better.By changing the ratio of the leaf transformation conditions,pre cultured for 3 d,the use of agro bacterium suspension diluted 30 times OD600 for 0.8 or 5 min,2 d after co culture,300 mg/L Cef had better effect,transformation.With the transformation of tobacco total DNA as template,the recombinant plasmid DNA as positive control,non transformed tobacco DNA as negative control,the use of Pycnoporus sanguineus laccase gene primers of PCR,results show that,the exogenous gene has been successfully integrated into tobacco.Transformation of tobacco leaf extract could be detected enzyme actinity,after purification,it is similar of Pycnoporus sanguineus laccase,and have ability of dye de-colorization.