Cloning, Sequencing And Evolution Analysis Of α-Amylase Genes From Xanthomonas Spp.

The study results of this paper can serve the two scientific subjects of our teaching and research group as basic data calculation and elementary exploration. The two subjects are: constructing high activity α-amylase genes by DNA shuffling technology and studying on the evolution in vitro by mutational PCR (with 5-Br-dUTP as substitute partly) and DNA shuffling technology.α-amylase (EC 3.2.1.1; 1,4-a-D-glucanohydrolase) can catalyzes the hydrolysis of α-1,4-glycosidic bonds of starch from middle and liberates α-maltose, α-glucose and α-limit dextrin stepwise. Therefore, a-amylase has been used widely in many industrial fields, such as glucose production, beer brewing, fermentation trade, textile industry, and so on.The study on a-amylase is one of the most active fields in enzyme industrial fields. With the development of biotechnology, more and more scientists and researchers attempt to use DNA shuffling technology to breed, screen and even "create" new a-amylase genes with higher activity and other new characters.The PCR primers used in this paper were designed based on the a-amylase gene sequences published in Genbank, and the two endonucleases, BamH I and Kpn I, were added to sequences of the upstream primer and downstream primer. Genomic DNAs were extracted from seven Xanthomonas spp. at first. Then eight a-amylase gene fragments were cloned with the genomic DNAs as templates by routine PCR. Following that, these gene fragments and plasmid vectors, pBluescript II KS+ and pUC18, were cut by BamH Iand Kpn I.The prepared insert DNA and vector DNA were linked by T4 DNA ligase. Then the linked products were transformed into the high competent cell of E. coli DH5a. Based on α-complementation of the detective β-galactosidase, positive recombinant clone were screened from X-gal plate. After recombinant DNAs were extracted, these DNAs were identified by single endonuclease cutting and two ones, functional identification and PCR identification.After sequencing and cutting vector sequences by Vecscreen software, these fragments were registered directly on-line and eight gi numbers were given: AY247106, AY220755, AY220754, AY216525, AY216526, AY220756, AY220753, AY220752, which are corresponding to zhyf001~zhyf008 respectively. These DNA fragments were aligned by the software, Vector NTI 6.0, with Neighbor Joining (NT) method. According to the alignment results, a genetree was constructed by Vector NTI 6.0.According to the analysis results, the following conclusions can be drawn:Firstly, these gene fragments are complete sequences except for zhyf008.Secondly, though several of these fragments are not α-amylase genes, there exists closer similarity between them and a-amylase genes.Thirdly, the evolutional relationship between AY220755 and AY216525, AY220754 is closest; Also does between AY220756 and AY167891, AY167890, AF428991, AE012174, AY165038; And closer between AY216526 and AE011710.