黑曲霉RAF106 sakA基因的敲除及功能分析

Gene knockout reveals the roles of sakA in Aspergillus niger RAF106

摘要:

【目的】 黑曲霉(Aspergillus niger) sakA基因是应激丝裂原活化蛋白激酶家族(mitogen-activated protein kinase, MAPK)的重要成员，然而，在黑曲霉中，关于SakA的分子功能研究很少。本研究通过构建黑曲霉sakA缺失突变株探究SakA在黑曲霉中的功能。【方法】 以黑曲霉RAF106为出发菌株，通过农杆菌介导法构建突变株ΔsakA菌株。监测ΔsakA菌株与野生菌株在3种不同培养基上生长与产孢情况；研究ΔsakA菌株对不同逆境胁迫的敏感性变化；测定ΔsakA菌株的胞内外淀粉酶、果胶酶和纤维素酶酶活差异；qRT-PCR分析ΔsakA菌株产孢相关基因、淀粉酶相关基因、果胶酶相关基因、纤维素酶相关基因及高渗调节相关基因的相对转录水平。【结果】 成功获得3株sakA缺失突变株ΔsakA菌株；发现ΔsakA菌株较野生菌株生长缓慢、产孢延迟及分生孢子梗分化延迟；ΔsakA菌株在0.6 mol/L KCl、0.8 mol/L NaCl和1.2 mol/L NaCl胁迫条件下，菌落生长均比野生型缓慢；ΔsakA菌株胞外淀粉酶产量提高20.68%−21.43%，胞内淀粉酶产量显著下降19.18%−20.26%；胞外果胶酶产量显著下降36.71%−38.30%，胞内果胶酶产量显著上升35.68%−36.53%；与野生菌株相比，ΔsakA菌株胞外纤维素酶产量显著下降28.04%−33.82%，而胞内纤维素酶产量显著上升15.28%−18.19%；产孢相关基因(fluG、sfgA、flbA、flbB、flbD、laeA、brlA、abaA、vosA、stuA和velB)的转录水平相较于野生菌株下调8.53%−90.87%；淀粉酶相关基因(amyC、amyD、amyE、amyF、amyG和amyH)及转录因子(amyR)下调8.87%−87.50%，果胶酶相关基因(aglB、lacA、pexB、pecA、pecC、pecB、endA、endC和poly)下调23.23%−84.01%，纤维素酶相关基因(xlnR、chbA、chbB和eglB)下调3.75%−81.02%，高渗调节相关基因(ena1、ena2、sho1、nik1、ypdl、pkA和hAD)下调5.27%−94.36%。【结论】 sakA基因正向调控黑曲霉RAF106的产孢能力，是产孢过程中的重要基因，其缺失影响黑曲霉分生孢子的产生；在参与渗透压胁迫应答的同时对淀粉酶、果胶酶与纤维素酶的合成与分泌有重要作用。

Abstract:

[Objective] The protein SakA encoded by sakA is a member of the mitogen-activated protein kinase (MAPK) family in Aspergillus niger. However, little is known about the roles of SakA in A. niger. In this study, we constructed the A. niger strains with knockout of sakA to investigate the roles of this gene. [Methods] The Agrobacterium-mediated method was utilized to construct ΔsakA strains from A. niger RAF106 (the wild type, WT). The growth and spore production of ΔsakA and WT were observed on three different media. The sensitivity of ΔsakA and WT to different stress conditions was studied. The intracellular and extracellular levels of amylase, pectinase, and cellulase were compared betweenΔsakA and WT. Real-time quantitative polymerase chain reaction (qRT-PCR) was employed to determine the relative transcript levels of the genes associated with spore production, amylase, pectinase, cellulase, and hyperosmotic regulation. [Results] Three ΔsakA strains were successfully obtained and verified by PCR and qRT-PCR. The ΔsakA strains had slow growth, delayed spore production, and delayed conidiophore differentiation compared with WT. The ΔsakA strains showcased slower colony growth than WT under the stress conditions of 0.6 mol/L KCl, 0.8 mol/L NaCl, and 1.2 mol/L NaCl. Compared with WT, the knockout of sakA increased the extracellular amylase production by 20.68%–21.43% and decreased the intracellular amylase production by 19.18%–20.26%, decreased the extracellular pectinase production by 36.71%–38.30% and increased the intracellular pectinase production by 35.68%–36.53%, decreased the extracellular cellulase production by 28.04%–33.82% and increased the intracellular cellulase production by 15.28%–18.19%. Compared with WT, the knockout of sakA down-regulated the transcript levels of spore production-related genes (fluG,sfgA, flbA, flbB, flbD, laeA, brlA, abaA,vosA, stuA, and velB) by 8.53%–90.87%. Furthermore, it down-regulated the transcript levels of amylase-related genes (amyC, amyD, amyE, amyF, amyG, and amyH) and the transcription factor (amyR) by 8.87%–87.50%, the pectinase-related genes (aglB, lacA,pexB, pecA, pecC, pecB,endA, endC, and poly) by 23.23%–84.01%, the cellulase-related genes (xlnR, chbA, chbB, and eglB) by 3.75%–81.02%, and the hyperosmotic regulation-related genes (ena1, ena2,sho1, nik1, ypdl, pkA, and hAD) by 5.27%–94.36%. [Conclusion] The sakA gene of A. niger positively regulates spore production and is essential for spore production. The knockout of sakA affects the spore production of A. niger. Furthermore, SakA plays a crucial role in the synthesis and secretion of amylase, pectinase, and cellulase as well as osmotic stress response.