摘要
组织蛋白酶在昆虫的消化、发育与变态中起重要作用,是捕食性蝽唾液(毒液)中普遍存在的成分,但其生理功能尚不清楚.本文利用同源比对的方法从蠋蝽Arma custos基因组中鉴定出组织蛋白酶基因,使用生物信息学软件分析其序列特征和进化关系,采用 RT-PCR 技术分析它们在成虫不同组织中的表达特征.结果表明,蠋蝽基因组中有 37个组织蛋白酶基因,根据结构域分为组织蛋白酶B(AcCAB1-4)、组织蛋白酶D(AcCAD1-13)和组织蛋白酶 L(AcCAL1-20).多序列比对及结构域预测结果表明,组织蛋白酶 B 和组织蛋白酶 L 均含有Peptidase_C1 结构域、保守的酶催化位点(谷氨酰胺、半胱氨酸、组氨酸和天冬酰胺)以及 1 个由半胱氨酸残基(C)和组氨酸残基(H)构成的催化二联体,而组织蛋白酶 D 中存在保守 Asp 结构域和 2 个保守的酶催化位点(天冬氨酸).RT-PCR结果显示,除AcCAL7和AcCAB2外,其余33个组织蛋白酶基因均在肠道中表达,且绝大多数表现特异性或高表达.AcCAL2、AcCAL5-7、AcCAL9等 20个组织蛋白酶基因在唾液腺中表达,且AcCAL2仅在主腺后叶和副腺表达,AcCAL7仅在主腺前叶表达,AcCAD13仅在主腺后叶表达,AcCAB2仅在主腺前叶和主腺后叶表达.这些结果表明,多数组织蛋白酶基因在蠋蝽口外消化和肠道消化中发挥消化功能.
Abstract
Cathepsins play crucial roles in the digestion,development,and metamorphosis of insects.They are commonly found components in the saliva(venom)of predatory bugs,while their physiological functions remain unclear.In this study,homology-based methods were employed to identify cathepsin genes from the genome of the stink bug Arma custos.Bioinformatics software was used to analyze their sequence characteristics and evolutionary relationships.RT-PCR technology was utilized to analyze their expression patterns in various adult tissues.The results revealed the presence of 37 protease genes in the A.custos genome,categorized into three groups based on structural domains:Cathepsin B(AcCAB1-4),cathepsin D(AcCAD1-13),and cathepsin L(AcCAL1-20).Multiple sequence alignment and domain prediction results indicated that cathepsin B and L both contain the Peptidase_C1 structural domain,conserved enzyme catalytic sites(glutamine,cysteine,histidine,and asparagine),and a catalytic dyad composed of cysteine(C)and histidine(H)residues.In contrast,cathepsin D possesses a conserved Asp domain and two conserved enzyme catalytic sites(aspartic acid).RT-PCR results demonstrated that,apart from AcCAL7 and AcCAB2,the remaining 33 protease genes were expressed in the gut,with the majority showing tissue-specific or high expression.Twenty cathepsin genes,including AcCAL2,AcCAL5-7,and AcCAL9,were expressed in the salivary glands,with AcCAL2 exclusively expressed in the main posterior gland and the accessory gland,AcCAL7 exclusively expressed in the main anterior gland,AcCAD13 exclusively expressed in the main gland posterior lobe,and AcCAB2 exclusively expressed in the main anterior and posterior glands.These findings suggest that multiple cathepsin genes play roles in external and gut digestion in A.custos.
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