In silico cloning and in vitro expression of a zinc binding dehydrogenase (GhZBDH) from upland cotton (Gossypium hirsutum)
1. Haide Institute of Tropical Agricultural Resources (HITAR), Sanya, 572025, China;
2. Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin, 150040, China;
3. Guizhou Key Lab of Agro-Bioengineering, College of Life Science, Guizhou University, Guiyang, 550025, China
Cotton Genomics and Genetics, 2010, Vol. 1, No. 2
Received: 10 Jul., 2010 Accepted: 15 Oct., 2010 Published: 30 Nov., 2010
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Many dehydrogenases in plants have an important structural zinc binding site, this Zn2+ plays an important role in enzyme structure and decisive role in protein stability. Our previous study had cloned a cDNA fragments Vdrg3, 409 bp in length, by using the approach of suppression subtractive hybridization, the cDNA was differentially expressed by induction of Verticillium dahliae toxin and has some homology with the Arabidopsis zinc-binding dehydrogenase. In present study we acquired two homological Gossypium EST sequences from cotton EST database and then spliced out a putative full-length cDNA sequence that contains a 1 299 bp open reading frame (ORF) based on in silico cloning strategy. According to this putative ORF sequence, we designed a pair of PCR primers as forward primer: 5'-CACCACTAGATCACAAGAATAATAATGG-3' and reverse primer: 5'-AAAAGGAGAAGCAATTTACATTATCTC-3', the cDNA with the length of 1 305 bp was amplified from the upland cotton (Gossypium hirsutum) that contains a complete ORF, encoding 434 amino acids.
By the analysis of Blast and multiple sequence alignment, we considered that the cDNA we cloned might belong to the zinc-binding dehydrogenase family, therefore named as GhZBDH (Gossypium hirsutum zinc-binding dehydrogenase). Conserved domain analysis showed that ADH C domain from 63 aa to 433 aa is similar as the third type of zinc-binding alcohol dehydrogenase ADH C domain for energy emerging and transformation, ADH_N domain locates from 87 aa to 221 aa and is ADH_N domain, NAD (P)-binding protein domain locates from 269 aa to 394 aa is NAD (P)-binding protein domain. Domain prediction results further suggested that this protein might have has a similar structure with similar to other species, which might play some similar roles of alcohol dehydrogenase.
We further ligated the cloned cotton zinc-binding dehydrogenase gene into the PQE-30 vector to make the recombinant vector as pQE 30-GhV3 then transformed into E. coli expression strain M15 by using the heat shock transformation approach. in vitro expression studies were carried out with the suitable temperature at 37â„ƒ, the optimal IPTG concentration of 0.1 mmol/L, and the optimal induction time of 3 hours, the inclusion proteins of 46 kD were expressed in E. coli and purified by using the method of Ni-NTA affinity chromatography. In conclusion, we suggested that GhZBDH be zinc-binding dehydrogenase, a response protein induced by toxin of Verticillium dahliae
Cotton (Gossypium hirsutum); zinc-binding dehydrogenase; GhZBDH; in silico cloning; in vitro expression
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