Abstract: 【Objective】This research was conducted to clone argininosuccinate synthetase gene from Gossypium hirsutum, obtain the fusion protein by prokaryotic expression system, detect the argininosuccinate synthetase activity of the fusion protein, and identify its effects on the growth, salt tolerance, L-citrulline (L-Cit) and L-arginine (L-Arg) content of engineering bacteria, aiming to lay a foundation for this gene’s function and mechanism. 【Method】The homologous cDNA fragment, named as GhASS1, was obtained from young leaves of cotton by the querying probe, a putative argininosuccinate synthetase cDNA sequence from Arabidopsis thaliana, in silico cloning and RT-PCR reaction. This fragment’s information was acquired by T/A cloning and sequencing. The genomic DNA and putative protein structure, function domain and homology were analyzed by bioinformatics software, and the phylogenetic tree was built. Its expression responses to salt tolerance were investigated by qRT-PCR. After the open reading frame of GhASS1 was linked to pCold-TF, the fusion expression vector, pCold-GhASS1, was constructed, and then transformed into the bacterial strain of Rosetta (DE3) plysS for expressing the recombination protein under the induction of IPTG. The molecular weight of recombination protein was tested by SDS-PAGE. The enzyme activities and specific activities were determinated by the method of pyrophosphoric fluorescence. The contents of free L-Arg and L-Cit in pCold-GhASS1 engineering bacteria were assayed by HPLC. The growth status of engineering bacteria was monitored under different NaCl concentrations in culture medium and different growth temperatures. The contents of free L-Arg and L-Cit, L-Arg/L-Cit ratio were compared. 【Result】 Sequencing analysis showed the genomic DNA of GhASS1 had 10 exons and 9 introns which was located on chromosome 1 of D genome in cotton, its cDNA fragment length was 1 584 bp containing 5′-untranslated region of 22 bp, the open reading frame of 1 485 bp and 3′- untranslated region of 77 bp, encoding 494 amino acid residues with a molecular mass of 54 kD and isoelectric point of 6.74. Sequence alignment analysis showed that GhASS1 shared 21.81%, 41.1% and 78.5% identity with the homologues from E. coli, yeast and Arabidopsis thaliana, respectively, and had the typical function domains of argininosuccinate synthetase. Phylogenetic analysis showed that GhASS1 was most closely related to the homologues from tomato, potato and tobacco, while was farthest to those from Picea sitchensis and Selaginellae moellendorfii. The expression of GhASS1 was up-regulated to the highest after 1 d under salt stress, then gradually declined in the leaves, indicating GhASS1 mRNA took part in the early response to salt tolerance. The SDS-PAGE analysis revealed that the fusion recombinant protein of GhASS1 was a molecular weight of 108 kD as expected. The fusion protein had argininosuccinate synthase activity in vitro. Compared with the control bacteria with pCold-TF, the contents of free L-Cit were decreased, while those of free L-Arg were increased in the engineering bacteria with pCold-GhASS1. In addition, the engineering bacteria with pCold-GhASS1 could be earlier into the logarithmic growth than the control, also showed stronger growth vigor and higher free L-Arg/L-Cit ratio under high NaCl contents in LB medium, revealing the engineering bacteria had stronger metabolic flow from L-Cit to L-Arg than the control. 【Conclusion】 It was the first report on the identification of argininosuccinate synthetase gene from cotton in plant. Its catalysis and physiology function were analyzed by the prokaryotic expression system, suggesting that it could be involved in the regulation of growth and ability of salt resistance in plant.

Key words: cotton (Gossypium hirsutum), argininosuccinate synthetase gene, salt tolerance, real-time PCR