【作者】 赵林；

【导师】 邓建新；

【作者基本信息】 长江大学 ， 植物保护， 2023， 硕士

【摘要】 马铃薯是全球第四大主要作物,仅次于水稻、小麦和玉米。目前,中国是世界上最大的马铃薯生产国,而马铃薯早疫病一直是其可持续生产的制约因素,其相关病原菌主要包括Alternaria solani、A.alternata、A.linariae、A.protenta、A.porri、A.grandis和A.blumeae等。2016年在湖北省宜昌市马铃薯早疫病叶上分离得到菌株YZU 161111,通过形态学、分子系统学和致病性检测将其鉴定为又一新病原,命名为宜昌链格孢（A.yichangensis）。枯草杆菌蛋白酶（Subtilisin）属丝氨酸蛋白水解酶类,广泛存在于细菌、真菌和寄生虫等生物体中。在植物病原真菌研究中,对枯草杆菌蛋白酶的研究较少。本课题组前期已对菌株YZU 161111进行了全基因组测序和转录组测序（菌丝和孢子）分析。本研究基于上述基因组数据,发现该菌包含13个枯草杆菌蛋白酶家族基因,并通过生物信息学方法,如基因染色体定位、蛋白质理化性质分析、蛋白质结构域分析、蛋白质二级结构分析、蛋白质三级结构分析、蛋白质信号肽预测、蛋白质跨膜结构域分析、基因表达量分析、蛋白序列系统发育树分析等,对其进行鉴定。基于同源性比对分析与系统发育树构建,选取EVM0006244基因（命名为AyPrb1）作为目的基因,通过同源重组技术成功构建基因敲除盒获得该基因缺失突变体（ΔAyPrb1）,并研究ΔAyPrb1在菌株生长发育、毒力等方面与野生型的差异性,探究该基因的功能。研究结果如下:生长表型检测结果表明,AyPrb1基因缺失突变体（ΔAyPrb1）与野生型相比,主要表现为气生菌丝生长缺陷,突变体菌丝分叉变多,生长弯曲;ΔAyPrb1分生孢子梗畸形,表现为分叉变多,整体粗大,孢子梗凹凸不平,产孢细胞膨大畸形;ΔAyPrb1分生孢子孢身细长、单喙（无双喙）,且在V8A和PCA培养基产孢量极少。致病性检测结果表明,ΔAyPrb1在马铃薯、番茄、茄子叶片致病力显著下降。穿透性试验检测与和黑色素含量检测发现,ΔAyPrb1穿透力明显下降以及黑色素含量也显著降低,且对细胞壁胁迫（SDS和CR）和氧化应激胁迫（H₂O₂）具有不同程度的敏感性。此外,该基因通过高渗透性甘油信号通路（High osmolarity glycerol,HOG）,调控宜昌链格孢对渗透胁迫的响应。在甘油含量检测时发现ΔAyPrb1中的甘油含量上升,而添加Na Cl处理后,ΔAyPrb1中的甘油含量上升更加显著,并伴随着脂滴的积累,其累计趋势与甘油一致。室内毒力测定结果表明,不论是野生型还是突变体ΔAyPrb1,6种供试药剂的抑菌作用较好,其中嘧菌酯毒力最大,喹啉铜毒力最小。ΔAyPrb1对于喹啉铜、咪鲜胺、戊唑醇、咯菌腈、嘧菌酯5种药剂敏感性升高,而对于氟啶胺药剂敏感性有所降低。从药剂作用机理方面来看,除嘧菌酯是抑制呼吸作用外,其余5种杀菌剂均是抑制病原菌丝生长,结果表明抑制病原菌呼吸作用的药剂开发比抑制菌丝生长和孢子形成的药剂开发更具应用前景。综上,枯草杆菌蛋白酶AyPrb1影响宜昌链格孢的生长发育与产孢;调控病菌穿透力以及黑色素的合成;参与调控细胞壁的完整性和氧化应激胁迫,负向调控HOG信号通路,从而调控甘油的合成和脂滴的代谢过程;还影响宜昌链格孢对杀菌剂的抗性,最终调控该菌的致病力。更多还原

【Abstract】 Potato(Solanum tuberosum L.)is the fourth largest staple crop after rice,wheat and maize worldwide.China is the largest potato producer worldwide currently.Early blight has been a limiting factor for sustainable production of potato.The pathogens of potato early blight mainly include Alternaria solani,A.alternata,A.linariae,A.protenta,A.porri,A.grandis,A.blumeae,etc.In 2016,strain YZU 161111 isolated from potato early blight leaf disease samples was collected in Yichang city,and was identified as a new pathogen(named A.yichangensis)based on morphological comparison,molecular identification and pathogenicity test.Subtilisines widely found in bacteria,fungi and parasites belong to a very diverse family of serine proteases.There are few reports on subtilisin in the study of plant pathogenic fungi.The whole genomic and transcriptomic(mycelium and spore)sequencing analysis of strain YZU 161111 had been carried out in the early stage.Based on the analysis of genomic data,13 subtilases were identified in the genome of A.yichangensis.Bioinformatics methods including chromosome localization,protein physical and chemical properties,protein domain analysis,protein secondary structure analysis,protein tertiary structure analysis,protein Signal P,transmembrane domain analysis,Micro Array or RNA-Seq and phylogenetic tree analysis of protein sequences were conducted.Based on homology comparison and phylogenetic analysis,EVM0006244 gene(named as AyPrb1)was selected as the target.Its knockout box was successfully constructed by homologous recombination technology and the deletion mutant of AyPrb1(ΔAyPrb1)was obtained.In order to determine the function,the differences between ΔAyPrb1 and the wild type strain were carried out in the growth,development,virulence and other aspects.The results were as following:Compared with the wild type,the phenotypes of ΔAyPrb1 were characterized by the growth deficiency of aerial hyphae.The mutant mycelia turned with many branching with curved growth.The conidiophores of ΔAyPrb1 were malformed with more branches,overall thickness and uneven wall.Its conidia were more elongated with single beak(without double beaks).And sporulation was very low in V8 A and PCA media.The pathogenicity of ΔAyPrb1 was significantly reduced in potato,tomato and eggplant leaves.The penetration and melanin content decreased obviously.Meanwhile,the results indicated that ΔAyPrb1 was sensitive to cell wall stress(SDS and CR)and oxidative stress(H2O2).Moreover,the gene regulated the response of A.yichangensis to osmotic stress by the high osmolarity glycerol(HOG)pathway.The glycerol concentration in ΔAyPrb1 increased,especially raised up when treated with Na Cl which accompanied with the emergence of lipid droplets.The accumulation trend of lipid droplets was consistent with that glycerol increase.In vitro,the results showed that the six tested fungicides were all great for the control management,of which azoxystrobin was the highest toxicity and oxine-copper was the lowest for both of the wild type and ΔAyPrb1.The sensitivity of ΔAyPrb1 to oxine-copper,prochloraz,tebuconazole,fludioxonil and azoxystrobin was upgraded,while the sensitivity to fluazinam downgraded.In addition,the other five fungicides inhibited the growth of mycelia except azoxystrobin which inhibited fungal respiration.The present results indicated that the development of fungicides for inhibiting fungal respiration had more application prospects than those for inhibiting mycelia growth and sporulation.In conclusion,the subtilis protease AyPrb1 affects the growth,development and sporulation of A.yichangensis,and it also regulates the penetration of pathogens and its synthesis of melanin.It not only participates in the regulation of cell wall integrity and oxidative stress,but also negatively regulates HOG signaling pathway which then resulted in the regulations of glycerol synthesis and lipid accumulation.Besides,it can also affect the resistance for fungicides.All in one,it ultimately leads to the reduction of virulence.更多还原