| All higher plants, many fungi and some protists have two mitochondrial electron transport pathways:a cytochrome pathway and an alternative pathway. The alternative pathway is also called anticyanide pathway which is cyanide insensitive. The alternative oxidase is the terminal oxidase in the alternative pathway. The alternative pathway is branched from the cytochrome pathway at ubiquinone pool. The alterantive oxidase transports the electron from ubiquinone to O2 and yield H2O without ATP produced.In this study, the ORF the alternative oxidase from Monascus ruber M-7(Mraoxl) and parts of its upstream and downstream sequence respectively were cloned by degenerate PCR and SON-PCR. The Mraoxl replacement vector was constructed and transformed into M. ruber M-7 by Agrobacterium tumefaciems mediated transformation. One Mraoxl deleted strain was obtained. The role of the Mraoxl played in the environmental stress and the effect of cAMP and citric acid on Mraoxl expression was analyzed. The results are as follows:1. Cloning and analysis of Mraoxl geneAccording to the reported amino acid sequence and nucleic acid sequence of alternative oxidase, a pair of degenerate primer was designed and cloned a fragment of Mraoxl. Based on the sequence achieved by degenerate PCR, the ORF of Mraoxl and part of its upstream and downstream sequence respectively were obtained by SON-PCR. The total sequence cloned was 4223bp. The ORF of alternative oxidase was 1198bp containing 3 exons and 2 introns and codes 350 amino acids. The upstream and downstream non-coding regions were 2329bp and 396bp respectively. And it was predicted that a CRE-like element and 2 zinc finger protein binding sites existed in the upstream of Mraoxl coding sequence.The amino acids sequence deduced from coding sequence showed high homologous with the alternative oxidases from other organisms and possessed the conservative characteristic domains of EXXH and EEE-Y which are related to the formation of which are related with the forming of Fe center. The 59 amino acid residues in the N-terminal were predicted to be the mitochondrial targeting sequence.2. Knock out of Mraoxl from M. rubber M-7The Mraoxl replacement vector was constructed using a hygromycin B resistance gene (hph) as selective marker and transformed into M. rubber M-7 by Agrobacterium tumefaciems mediated transformation.205 transformants were obtained by twice transformation of M. rubber M-7. And one Mraoxl deleted mutant (ΔMraoxl) was achieved after the identification by PCR and southern blotting.3 Primary studies for the function of Mraoxl gene3.1 Effects of H2O2, high temperature, pH and osmotic pressure on the conidia viability ofΔMraoxl and M-7In order to identify if the Mraoxl gene particapated in the resistance to environmental stress,105/mL conidia liquid ofΔMraoxl and M-7 was exposed to H2O2 Solution, high temperature water, citric acid-dibasic sodium phosphate buffers of differ pH and high concentration NaCl solution, and the conidia viabilities were analyzed after these treatments.After the exposition to 10,20 and 30mmol/L H2O2 solution for 5 hours, the conidia viabilities ofΔMraoxl were lower than M-7 significantly. After exposition to water of 40 and 50℃, the conidia of M-7 andΔMraoxl showed a decrease and difference in the percentage of viabilities, and the percentage of viability conidia ofΔMraoxl were 18% lower than that of M-7, which disparity was the most significant, when the conidia were exposed in water of 40℃for 2h. The conidia viability of M-7 andΔMraoxl showed 29%lower than that of M-7, after being exposed to citric acid-dibasic sodium phosphate buffer of 8.0 for 4h. These results indicated that the Mraoxl gene play a role in response to environmental stress.3.2 Effects of cAMP and citric acid on the growing ofΔMraoxl and M-7The addition of 2mM cAMP and 8mM citric acid to PDA medium can significantly accelerated the growth of M-7 at the early stage, while citric acid and cAMP hadn't the same effect on AMraoxl. It seems that cAMP and citric acid can enhance the expression of Mraox1 at the early growth stage.
|
PDF Full Text Request