Directed Evolution Of E.coli Beta-galactosidase In Vitro And Agrobacterium-mediated Transformation Of Malus With DREB1A Gene

E.coli beta-galactosidase, encoded by LacZ gene, is widely used as reporter gene which was used to screen the transformants in genetic transformation.In this study, LacZ from E.coli was evolved by DNA shuffling . Three rounds of DNA shuffling and colony screening on high-throughput were performed. Evolved LacZ gene has been attained. Compared with native LacZ, the nucleotide sequence of the evolved LacZ gene showed 13 base changes, resulting in 9 amino acid changes from the native enzyme. The purified mutant enzyme showed a 3-fold increasing in o-nitrophenyl-galactopyanoside specific activity.DREB1A gene is a trans-acting factor in Arabidopsis. Its protein interacts with a 9-base pair(bp) conserved sequence (TACCGACAT) of DUE, which is an essential cis-acting element for regulation in response to dehydration and cold stress.In the article, total RNA was isolated from Arabidopsis, and single stranded cDNA was synthesized. Then the two nucleotides of DreBlAZl and DreBlAFl were used to prime the synthesis and amplification of DREB1A gene by PCR. After the PCR product was cloned into the TA vector, nucleotide sequence was determined. The result of DNA sequence analysis indicated that the nucleotide sequence of cloned DREB1A gene is same with the sequence encoded in Genbank. Binary vector pDreB1A including DREB1A gene controlled by double CaMV35S promoter was constructed. The pDreB1 A was transformed into Agrobacterium tumefaciens strain LBA4404 by electroporation .An efficient Agrobacterium-mediated regeneration and transformation system of apple was developed. Using advanced regeneration system(MS + BA 4 mg/L + NAA 0.1 mg/L), regeneration rate can reach 100%. The DREB1A gene were introduced into apple(Malus micromalus Makino) by Agrobacterium-mediated transformation. The transgenic plant is hopeful to improve the production and reduce the limitation of plant domain.