Cloning And Analysis Of The Transcriptional Regulator Factor Gene AreA?nmrA From Pleurotus Ostreatus

Pleurotus ostreatus is the most widely cultivated edible fungus in China.Nitrogen,carbon,water,mineral,growth factor and appropreiate C:N ratio are needed for growth and development of P.ostreatus.As the major nutrient element for P.ostreatus,nitrogen element plays a crucial role during its growth and development.However,the study on the reglation of nitrogen metabolism in P.ostreatus is still blank.In this study,the GO annotations and sequences of areA and nmrA gene from P.ostreatus PC 15 were obtained through a genome-wide serch.Then,areA and nmrA gene from P.ostreatus New831 was coloned and studied,including the following aspects: the CDS fragemt of these two genes were obtained by using P.ostreatus New831 c DNA as the template;the basic traits of these two genes and its deduced proteins were further analyzed by bioinformatics analysis;heterologous expression and SDS-PGAE analysis of areA and nmrA gene were performed;Relative expression level of areA,nmrA060 at different nitrogen source of P.ostreatus;the interaction between AreA protein and NmrA protein was studied via yeast two-hybrid assays;the overexpression and antisense silencing vectors for areA and nmrA gene were constructed,which laid foundation for further study of areA and nmrA gene.The main results of this study are as follows:1.Effects of different nitrogen forms on the mycelial growth of P.ostreatus were studied by seperatly using NH₄⁺-N?L-Glu?L-Gln?NO₃^--N?urea and L-Arg as the sole nitrogen source.The results showed that mycelia grew better on the NH₄⁺-N?L-Glu?L-Gln media than on the NO₃^--N?urea and L-Arg media.2.The CDS regions of areA,nmrA056 and nmrA060 were successfully cloned by using c DNA as the template.The results showed that the length of areA is 3,066 bp,encoding 1,021 amino acids.The length of nmrA056 is 1,083 bp,encoding 360 amino acids.The length of nmrA060 is 1,146 bp,encoding 382 amino acids.3.The gene structure of areA,nmrA056 and nmrA060,the subcellular localization and domains of AreA,NmrA056 and NmrA060 were seperatly predicted by bioinformatics analysis.The results showed that areA contains 5 introns,6 exons and 3 'UTR.A NLS is located at 585-608 region of AreA,and the subcellular localization prediction result also indicates AreA protein is localized in nucleus.A conserved zinc finger domain(C-X₂-C-X₁₇-C-X₂-C)was found at the C-terminus of AreA protein.nmrA056 and nmrA060 gene all contain 3 introns and 4 exons.NmrA056 and NmrA060 were predicted localized in cytoplasm.A NADP binding site and a regulatory binding site were found in NmrA056 and NmrA060,which indicates that these two genes may play a role in transcription regulation.4.The herologous expression vectors of areA₁₅₆₆,nmrA056,nmrA060 were successfully constructed,then expression of AreA1566,NmrA056,NmrA060 protein were induced by IPTG.The SDS-PAGE results showed that the molecular weight pf AreA1566,NmrA056,NmrA060 is 54 k Da,40 k Da,43 k Da,respectively.5.Relative expression level of areA,nmrA060 at different nitrogen source of P.ostreatus that areA was relatively high in secondary nitrogen sources;nmrA060 gene was relatively high in preferential nitrogen sources.6.The yest two-hyrid vectors of areA₁₅₆₆,nmrA056,nmrA060 were successfully constructed,and the yeast two-hybrid experiments were conducted.The primary results showed that there are no interactions between AreA and NmrA056 or Nmr060.7.The overexpression and antisense silencing vectors for areA₁₅₆₆,nmrA056 were successfully constructed.And then the A.tumefaciens mediated transformation was conducted.Some putative transformants have been obtained at present.