摘要
【目的】丰富蜜蜂球囊菌(Ascosphaera apis)几丁质酶3(AaCHIT3)的理化性质和分子特征信息,为AaCHIT3基因的功能研究提供科学依据。【方法】利用Expasy网站的ProtParam和ProtScale软件分别预测AaCHIT3蛋白的理化性质和亲水性,采用MEME软件鉴定保守基序和结构域,通过Mega 11.0软件对蜜蜂球囊菌及其他真菌的CHIT3进行系统进化分析。使用RT-qPCR检测蜜蜂球囊菌侵染意大利蜜蜂(Apis mellifera ligustica)工蜂幼虫过程中AaCHIT3的表达模式。【结果】AaCHIT3由372个氨基酸组成,分子质量约为43.03 ku,理论等电点为5.05,分子式为C_(1953)H_(2986)N_(514)O_(559)S_(13),平均亲水系数为-0.39。AaCHIT3中,α-螺旋含102个氨基酸,延长链由53个氨基酸组成,无规则卷曲结构由217个氨基酸构成。AaCHIT3的三级结构与模板A0A168BB95.1.A的序列一致性达100%。AaCHIT3包含2个结构域(GH18和CCDC50),而疏棉状嗜热丝孢菌(Thermomyces lanuginosus)、石膏样奈尼兹皮真菌(Nannizzia gypsea)等其他9种真菌的CHIT3仅含有1个相同结构域(GH18-CTS3)。AaCHIT3含有7个保守基序,疏棉状嗜热丝孢菌、奥菲迪菌(Ophidiomyces ophidiicola)、石膏样奈尼兹皮真菌、副球孢子菌(Paracoccidioides lutzii)和印度块菌(Tuber indicum)的CHIT3均包含9个相同保守基序,曲霉菌(Aspergillus hiratsukae)、烟曲霉(Aspergillus fumigatus)、灰青霉(Penicillium canescens)和紫红曲霉(Monascus purpureus)包含10个相同保守基序。系统进化分析显示,蜜蜂球囊菌和印度块菌的CHIT3在进化树上各自单独为一支。与蜜蜂球囊菌接种后1 d(1 dpi)相比,AaCHIT3的表达水平在2 dpi显著上调,3 dpi时则下调。【结论】AaCHIT3是一种潜在的亲水性蛋白;蜜蜂球囊菌与上述其他真菌的CHIT3在保守性和同源性方面均较低。在蜜蜂球囊菌侵染意大利蜜蜂工蜂幼虫过程中,AaCHIT3的表达呈现先上调后下调的趋势,提示AaCHIT3可能在蜜蜂球囊菌的侵染过程中发挥重要作用。
【Objective】This study aimed to enrich the knowledge of the physicochemical and molecular characteristics of chitinase 3 in Ascosphaera apis (AaCHIT3) and offer a scientific basis for functional study in AaCHIT3 gene. 【Method】The physicochemical properties of the AaCHIT3 protein were predicted using ProtParam on Expasy website, together with the analysis on its hydrophilicity via ProtScale software. Then the conserved motifs and domains of AaCHIT3 were identified using MEME software, and the phylogenetic analysis of CHIT3 from A.apis and other fungi was conducted using Mega 11.0 software. Additionally, the expression pattern of AaCHIT3 during the infection of A.apis on Apis mellifera ligustica worker larvae was examined through RT-qPCR. 【Result】AaCHIT3 contained 372 amino acids, with a molecular weight of approximately 43.03 ku, a theoretical isoelectric point of 5.05, molecular formula of C_(1953)H_(2986)N_(514)O_(559)S_(13), and average hydrophilicity coefficient of -0.39. In AaCHIT3, 102 amino acids formed α-helices, 53 amino acids constituted extended strands, and 217 amino acids formed random coils. The tertiary structure of AaCHIT3 shared 100% sequence homology with template A0A168BB95.1.A. AaCHIT3 had 2 structural domains (GH18 and CCDC50), while CHIT3 of the other 9 fungi such as Thermomyces lanuginosus and Nannizzia gypsea only contained only 1 identical structural domain (GH18-CTS3). AaCHIT3 harbored 7 conserved motifs, while CHIT3 from other 5 fungi like T.lanuginosus, Ophidiomyces ophidiicola, N.gypsea, Paracoccidioides lutzii and Tuber indicum had 9 identical ones, and CHIT3 from Aspergillus hiratsukae, Aspergillus fumigatus, Penicillium canescens and M.purpureus shared 10 identical conserved motifs. CHIT3 from A.apis and T.indicum formed separate clade on the phylogenetic tree. In comparison to the level at 1 day post inoculation (1 dpi) with A.apis, the expression level of AaCHIT3 was significantly upregulated at 2 dpi while downregulated at 3 dpi. 【Conclusion】AaCHIT3 is a potentially hydrophilic protein, and the homology and conservation of CHIT3 between A.apis and other fungal species mentioned above is weak;during A.apis's infection on A.m.ligustica worker larvae, the expression of AaCHIT3 is initially activated and then inhibited, implying the potential function of AaCHIT3 in the infection process of A.apis.
作者
王梦怡
吴陶
范小雪
胡艳雯
王炜
郑一荻
邱剑丰
张荣华
严提珍
陈大福
郭睿
WANG Mengyi;WU Tao;FAN Xiaoxue;HU Yanwen;WANG Wei;ZHENG Yidi;QIU Jianfeng;ZHANG Ronghua;YAN Tizhen;CHEN Dafu;GUO Rui;Unit:(College of Bee Science and Biomedicine,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;National and Local Junited Engineering Laboratory of Natural Biotoxin,Fuzhou,Fujian 350002,China;Apitherapy Research Institute,Fuzhou,Fujian 350002,China;Dongguan Maternal and Children Health Hospital,Dongguan,Guangdong 523000,China)
出处
《福建农林大学学报(自然科学版)》
北大核心
2025年第5期640-646,共7页
Journal of Fujian Agriculture and Forestry University:Natural Science Edition
基金
国家自然科学基金面上项目(32372943、32172792)
国家现代农业产业技术体系建设专项资金(蜜蜂)(CARS-4-KXJ7)
福建省自然科学基金面上项目(2022J01133)
福建农林大学硕士生导师团队项目(郭睿)
福建农林大学科技创新专项基金(KFb22060XA)
福建省大学生创新创业训练计划项目(S202410389062、S202410389064、S202410389056)。
关键词
蜜蜂白垩病
蜜蜂球囊菌
意大利蜜蜂
几丁质
几丁质酶
分子特征
表达模式
bee chalkbrood disease
Ascosphaera apis
Apis mellifera ligustica
chitin
chitinase
molecular characteristics
expression pattern
