Studies On The Cloning Of Halo-tolerant-related Genes From Eukaryote And Procaryote

According to the essential character that no intron can be found in the genes of prokaryote, a pair of primer has been designed on the basis of the conserved sequence of halo-tolerant-related mtlD (mannitol-1 -phosphate dehydogenase) gene in Escherichia coli. The mtlD gene of halophilous Pseudomonas sp.cn 4902 has been copied by the PCR reaction of making use of its genomic DNA as template and inserted into pMDl 8-T vector. Sequence of this gene shows that it is 1149 bp in length and codes for 383 amino acids. The nucleotide sequence and deduced amino acid of this gene share more than 90% homologies with mtlD gene of other organisms, especially for the mtlD gene from E.coli K12 that reaches as high as 99%. Furthermore this gene was subcloned to construct the high expression recombinant vector pBH and transferred into E.coli JM101. The result of protein SDS-PAGE electrophoresis of transformant indicated that a 41 kDa protein coincided with the molecular weight of mtlD gene transferred, which was 6.7% of the total protein, had been induced in this bacterium under 42 ℃. As the result, the tolerant level against to NaCl of E.coli JM101 transferred has raised from 0.9 to 1.1 mol/L, 22% higher than control. Furthermore, in order to transferring the gene to crop plant for expression, we constructed the recombinant plasmid pCH1301 for expression in plant and transferred it successfully into Agrobacterium rhizogenes EHA105 .Because of the property of unsuccessive gene (split gene) in eukaryote, in order to cloning the halo-tolerant-related genes, a cDNA library of halophilic Dunaliella bardawill (green alga) which withstands the high level osmotic shocks has been constructed on the basis of plasmid pUCl 9 and its host E.coli JM101. Two primers (Pi and Pj) were designed according to the 424bp cDNA fragment of Dunaliella bardawill ATP synthase alpha chain gene found by DDRT-PCR in our lab. Then the sequencing primers (positive and negative) near the multiple cloning site on pUC19 were matched for PI and ?2 respectively to form two pairs of primers, with the cDNA-pUC19 ligations as template in PCR amplification reactions. Three bands of about 800bp, 600bp and SOObp in size has been gained after PCR for two periods, and subsequently cloned into vector pMC18-T for sequencing. At the same time, the size of foreign cDNA fragments inserted in the cDNA library has been determined between 1.1 to 2 3kb The efficiency of cloning of the library was 2.36xl05 pfu cDNA, with 96% of the reconstructed ratio In sum, the cDNA library consists of almost of the mRNA copies in Dunaliella bardawill and is suitable to screen most cDNA clone.We have established an easy way for cloning structural genes by the combination of polymerase chain reaction (PCR) and cDNA library. As compared with others, thismethod for cloning genes is easy and efficient. We can conveniently gain morehalo-tolerant-related gene(s) from the cDNA library of Dunaliella bardawill by thisway.