اثر سرب بر مقدار گلومالین هیفی و ریشه ‏ای واکنش ‏پذیر با آنتی ‏بادی مونوکلونال و بردفورد در شرایط کشت درون شیشه‏ ای و گلدانی

گلومالین به عنوان گلیکوپروتئین شناخته شده در قارچ‏های میکوریزی شاخه گلومرومایکوتا و راسته گلومرال با دو روش بردفورد و آنتی‏بادی مونوکلونال اندازه‏گیری می‏شود. با این پیش فرض که تنش ناشی از فلز سمی منجر به افزایش بیان و تولید گلومالین به عنوان پروتئین شوک حرارتی می‏گردد، پژوهشی جهت مطالعه و مقایسه مقدار گلومالین تولیدی در شرایط کشت گلدانی در همزیستی قارچ رایزوفاگوس ایرگولاریز با گیاه شبدر سفید (trifolium repens l.) و کشت درون شیشه‏ای در همزیستی ریشه‏های تراریخت هویج (daucus carota l.) با همان قارچ تحت تنش سرب طراحی گردید. در کشت درون شیشه‏ای با افزایش غلظت سرب (0، 01/0، 1/0 و 1 میلی‏مولار pb+2) مجموع درصد فراوانی هیف و اسپور کاهش یافت درحالی که پروتئین واکنش‏پذیر بردفورد و پروتئین واکنش پذیر با آنتی بادی در بخش هیفی افزایش یافت. در کشت گلدانی با افزایش غلظت سرب (0، 150، 300 و 450 میکرومولار pb+2) درصد طول ریشه‏ کلنی‏شده نسبت به شاهد افزایش نشان داد. به طور کلی با افزایش غلظت سرب، پروتئین واکنش‏پذیر بردفورد و پروتئین واکنش پذیر با آنتی‏بادی در بخش هیفی و ریشه‏ای کشت گلدانی افزایش یافت. آنتی بادی مونوکلونال واکنش متقاطع ناچیزی با پروتئین‏های ریشه‏های غیرمیکوریزی نشان داد. بنابراین تولید گلومالین از طریق ممانعت از تغییر شکل پروتئین‏های مهم و حیاتی گیاه به عنوان پروتئین شوک حرارتی، ساز و کار حفاظتی قارچ های میکوریز آربوسکولار در همزیستی با گیاه برای کاهش تنش ناشی از سرب می‏باشد.

The Effect of Lead on the Glomalin Content of Hypha and Root Reactive with Monoclonal Antibody and Bradford in both in Vitro and Pot Culture Conditions

Introduction: Glomalin is known as a specific fungal glycoprotein belonging to the order Glomerales in phylum Glomeromycota and has been introduced as a heat shock protein. We hypothesised that increasing the level of Pb would lead to increase in glomalin production. Glomalin is usually determined by two methods, the Bradford protein dyebinding assay and the enzymelinked immunosorbent assay (ELISA). Since many laboratories are not equipped to carry out the ELISA assay, many studies have measured glomalinrelated soil protein using the Bradford colorimetric total protein assay. While, the ELISA method specifically measures glomalin by using monoclonal antibody MAb32B11. Materials and Methods: The pot experiment was conducted in the sterile freeglomalin sand with Trifolium repens L. mycorrhized by Rhizophagus irregularis fungus and treated with the Pb levels of 0, 150, 300 and 450 µM. Thus, in vitro experiment was performed in twocompartments plates containing of the transformed carrot roots (Daucus carota L.) mycorrhized with the same fungus in root compartment and hyphal compartment treated with the Pb levels of 0, 0.01, 0.1 and 1 mM as Pb(NO3)2. For glomalin extraction, hyphal or root samples were autoclaved at 121 ⁰C with 50 mM sodium citrate buffer for 60 min in three cycles. Protein concentrations in the extracted samples were determined using a modified Bradford protein assay. Also, glomalin content in the samples were determined by indirect ELISA using monoclonal antibody MAb32B11. The percentages of the total root length were colonised by mycorrhizal fungi in pot culture and both hyphal and spore densities in the metalcontaining hyphal compartment were determined. Results and Discussion: In the in vitro culture the percentage of total hyphae and spore frequency decreased, while Bradford reactive total hyphal protein (BRHP) and Immunoreactive hyphal protein (IRHP) in hyphal compartment increased as the concentrations of Pb increased. Also, there was positive and significant correlation between immunoreactive hyphal protein (IRHP) and Bradford reactive total hyphal protein (BRHP) in hyphal compartment of in vitro culture (r= 0.941**). In the pot culture, the percentage of the total mycorrhized root length in all the treatments increased compared to the unleaded control as the concentrations of Pb raised. In general, Bradford reactive total protein and Immunoreactive protein in both the hyphal and root compartments of pot culture increased with increasing the Pb levels. Also, there were positive and significant correlations between immunoreactive hyphal protein (IRHP) with Bradford reactive total hyphal protein (BRHP) (r= 0.845 **) and immunoreactive root protein (IRRP) with Bradford reactive total root protein (BRRP) (r= 0.706 **) in pot experiment. Some previously researches had reported correlation between ELISA with Bradford contents, whether, Bradford and ELISA values were nearly the same, this means that the extraction process mostly separates glomalin. The results of nonmycorrhizal roots indicated that a small proportion of root protein is crossreactive with the MAb32B11 antibody. There are some evidences that MAb32B11 is slightly crossreactive with plant compounds, nonAMF species, and nontarget proteins present in large concentration, such as BSA. Additionally, we found the increasing of BRRP contents of AMFcolonized root compared to the nonmycorhizal roots. This may be as a result of uptake and storage of arginine within AMF intraradical hyphae. Considering IRHP to BRHP ratio indicates that immunoreactivity percentage enhances by rising Pb levels. Immunoreactivity indicates a molecular configuration similar to the configuration of glomalin on hyphae. Our findings are in agreement with previous observations confirming that the toxicityinduced stress by metals may be enhancing glomalin production by AMF. The sequence of the glomalin gene revealed that is likely a 60KDa heatshock protein (Hsp) homolog. Glomalin relation with the heat shock proteins clarifies how stress is imposed by heavy metals may rapidly increase glomalin production by AMF and its concentrations in polluted soils. Conclusion: The high contents of glomalin along with the increasing of Pb concentrations may be explained by the overexpression of this protein. This suggests that under Pbinduced stress and the toxic effect of Pb, the fungus exerts a protective mechanism against toxicant. Therefore, glomalin as a heat shock protein can involve in the reduction of possible cytosolic damages and the transfiguration of proteins under Pb toxicity. We can conclude that glomalin may reduce toxic elements availability via their stabilization and decrease their toxicity risk to other microorganisms and plants in heavy metal polluted sites.