前纤维蛋白点突变对粗糙脉孢菌生长的影响及其生化机制研究

Effects of point-mutated profilins on the growth of Neurosporacrassa and the biochemical mechanisms

摘要:

【目的】为进一步了解前纤维蛋白(profilin，PFN)在丝状真菌中的功能，本文以粗糙脉孢菌(Neurospora crassa)为研究对象，进行了前纤维蛋白对其菌落生长和肌动蛋白(actin)聚合特性影响的探究。【方法】通过采用定点突变、同源重组、分生孢子过膜和PCR等技术，获得粗糙脉孢菌前纤维蛋白F78(F78A和F78D)和V113(V113E、V113R和V113W)的点突变体。利用平板生长法、竞争性生长管和显微镜观察检测表型变化，并结合多聚脯氨酸亲和层析纯化、荧光分光光度技术和高速共沉淀等技术分析点突变的前纤维蛋白对肌动蛋白聚合特性的影响。【结果】获得的粗糙脉孢菌前纤维蛋白点突变株F78A、F78D、V113E、V113R和V113W，与对照菌株ku70^RIP相比，突变株生长均明显减慢(P<0.05)，其中PFN (F78D)和PFN (V113W)的突变体在生长的12–48 h，菌落直径分别仅为对照的20.0%–75.7%和12.7%–39.2%。竞争性生长管分析表明，PFN (F78D)和PFN (V113W)突变株菌丝生长速度受到显著抑制，分生孢子形成的节律并未发生明显改变。将纯化获得的前纤维蛋白野生型及点突变株F78D和V113W蛋白进行肌动蛋白的成核研究发现，前纤维蛋白能抑制肌动蛋白自发的成核过程，并表现为浓度依赖效应；而点突变蛋白F78D和V113W抑制肌动蛋白自发成核的作用减弱；进一步对肌动蛋白聚合特性的研究发现：野生型的前纤维蛋白在0–5 mmol/L范围内可抑制肌动蛋白的聚合，且随浓度上升抑制作用进一步增强，致使上清中单体肌动蛋白含量最高可上升至82%左右；而5 mmol/L点突变蛋白F78D和V113W抑制肌动蛋白聚合的作用均明显减弱，分别使单体肌动蛋白的含量比对照下降12.0%(P<0.01)和30.7%(P<0.01)。【结论】本研究证实前纤维蛋白在粗糙脉孢菌中起着重要的作用，其F78和V113是重要的活性位点，对于调节肌动蛋白的聚合解聚过程和粗糙脉孢菌的生长发育具有重要作用。

Abstract:

[Objective]In order to further clarify the functions of profilin (PFN) in filamentous fungi, we explored the effects of point-mutated PFNs on the colony growth and actin polymerization of Neurospora crassa. [Methods] The PFN F78 mutants (F78A and F78D) and V113 mutants (V113E, V113R and V113W) of N.crassa were obtained by techniques of site-directed mutagenesis, homologous recombination, filtration of conidia and PCR. With plate method, race tube assay and microscopy, the phenotypic changes of colonies were observed, and together with polyproline affinity chromatography, fluorescence spectrophotometry and high-speed co-sedimentation, the effect of point mutation of PFN on the actin nucleation and polymerization was analyzed. [Results] Mutants F78A, F78D, V113E, V113R, and V113W all grew slowly compared with the control strain ku70^RIP (P<0.05). Particularly, the colony diameters of F78D and V113W were only 20%–75.7% and 12.7%–39.2% that of the control at 12–48 h. Race tube analysis demonstrated that mycelia of F78D and V113W presented slow growth in comparison with the control. However, both of them exhibited near-normal conidiation rhythms. In addition, the purified wild-type PFN could inhibit the spontaneous nucleation of actin in a concentration-dependent manner, while the inhibition of mutant proteins PFN (F78D) and PFN (V113W) on the actin nucleation was weakened. Wild-type PFN could suppress the polymerization of actin in the concentration range of 0–5 mmol/L, and the suppression was enhanced with the increase of the concentration, causing the monomer actin content in the supernatant to peak at about 82%. However, the inhibitory effect of 5 mmol/L PFN (F78D) and PFN (V113W) on actin polymerization was significantly weakened, as manifested by the decrease of actin monomer content by 12.0% (P<0.01) and 30.7% (P<0.01), respectively, from the control. [Conclusion]This study confirmed that PFN played an important role in N.crassa, and F78 and V113 were important active sites for regulating the polymerization and depolymerization of actin and the growth and development of N.crassa.