人工合成抗菌肽对棉花黄萎病菌的抑菌效果

Inhibitory activities of the synthetic antimicrobial peptides against cotton Verticillium dahliae

大丽轮枝菌(Verticillium dahliae)是引起棉花黄萎病的真菌病害,广泛存在于中国的产棉区,使棉花产量和品质大幅下降。虽然,人们长期以来一直在寻找抗病基因,但很难在陆地棉中发现。为了寻找对棉花黄萎病菌有强抑制作用的抗菌肽或基因,人工设计和合成了20条抗菌肽(简称"人工肽"),通过抑菌圈法从中筛选到3条最有潜力的人工肽(D4E1、D51和D28),并与2条天然抗菌肽(Decoralin和Pseudin,简称"天然肽")进行对比,用于鉴定其对棉花黄萎病菌的抑菌活性。结果表明,D4E1和D51这2条人工肽的抑菌活性最强,在多肽质量浓度为20μg/mL时,几乎能100%地抑制黄萎病菌的菌丝生长和孢子萌发,显示出高抗特性。同时,实验采用生物信息学软件和反相高效液相色谱法测定了抗菌肽疏水活性,结果表明反相高效液相色谱保留时间可以更有效地表示抗菌肽的疏水活性,从而能更准确推测抗菌肽的抑菌活性。上述研究结果说明:1)如果有一个好的人工设计,人工肽对黄萎病菌的抑制效果会比天然肽更佳;2)人工肽的氨基酸序列可推导出核苷酸序列,作为抗棉花黄萎病的候选基因,用转基因育种技术可获得抗病新品种。

Summary Verticillium wilt, caused by Verticillium dahliae （VD）, is the most disastrous pathogen in cotton fungal diseases, which causes huge monetary losses by inducing vascular wilt and exists widely in China cotton planting area. For a long time, scientists are engaged for identifying resistant genes against this pathogen, but can not find any one in tetraploid cotton varieties （Gossypiurn hirsutum）. However, antimicrobial peptide can be chosen as an ideal candidate because of its good features of broad- spectrum antibacterial activity, strong thermal stability and immunogenicity, and small molecular weight. Therefore, 20 novel antimicrobial peptides were designed and synthesized to analyze their inhibitory activities against the VD pathogen. After the preliminary screening, 3 peptides （D4E1, DS1, D28） were selected out from 20 synthetic peptides and were used to compare with 2 natural antimicrobial peptides （Decoralin, Pseudin）. Among these 5 peptides, two synthetic peptides, D4E1 and DS1, showed stronger resistance than the naturalpeptides, which could inhibit the growth of E. coli and V. dahliae and even kill 100% of conidia at very low peptide concentration, such as 20μg/mL. The reason for this huge difference might he hydrophobicity of antimicrobial peptides which had been recognized as one of the major parameters influencing the antimicrobial activity of peptides. Two methods, Protein Identification and Analysis Tools on the ExPASy server and Reverse Phase High Performance Liquid Chromatography （RP HPLC）, were used to determine their mean hydrophobicity of these peptides. The result revealed that the RP HPLC retention time could be better to understand the relationship between the peptide hydrophobicity and their antimicrobial activities. This study suggested that the synthetic peptides may be stronger in antimicrobial activity than the natural peptides if a good design were taken, and that the amino acid sequence of synthetic peptides with ideal activity, such as D4E1 and D51 in this research, can be deduced into the nucleotide sequence which could be an effective resistant gone against the cotton wilt disease in the transgenic cotton developed by the genetic engineering.