家蚕丝氨酸蛋白酶BmHP14基因的克隆与表达模式分析

家蚕受到外源微生物侵染或损伤时,前酚氧化酶原级联反应中的起始丝氨酸蛋白酶会激活下游信号通路,最终产生黑色素。采用RACE技术,获得了家蚕前酚氧化酶原级联反应起始丝氨酸蛋白酶——血淋巴蛋白酶编码基因的全长cDNA序列,命名为BmHP14(GenBank登录号:JQ954757)。BmHP14 cDNA全长2 508 bp,开放阅读框为2 013 bp,编码670个氨基酸,预测蛋白质分子质量71 kD,等电点5.09,N端17个氨基酸预测为信号肽序列。多重序列比对显示BmHP14与烟草天蛾HP14的相似度很高,达到57%;分子进化树中二者也聚为一支。RT-PCR分析表明,BmHP14在家蚕5龄第3天幼虫脂肪体、马氏管、精巢、卵巢、表皮、血细胞、头部均有表达,其中以脂肪体中的表达水平最高。以黑胸败血芽孢杆菌、大肠杆菌、球孢白僵菌注射侵染家蚕5龄第3天幼虫,Real-time PCR检测显示在受到病菌侵染后,幼虫脂肪体中的BmHP14表达上调。Westernblotting检测结果显示,BmHP14在家蚕体液中以前体和成熟体的形式共同存在。研究结果提示,BmHP14是家蚕前酚氧化酶原级联反应信号通路中的关键酶。

Gene Cloning and Expression Profile Analysis of Serine Protease BmHP14 in the Silkworm,Bombyx mori

When the silkworm(Bombyx mori) is confronted with invasion of microorganisms or injuries,serine proteases in the prophenoloxidase(proPO) cascade will start the activation of downstream signals,resulting in the generation of melanin.Using RACE technology,we cloned the full-length cDNA of a serine protease(hemolymph proteinase) initiating the proPO cascade of silkworm and named it as BmHP14(GenBank accession No.JQ954757).The full-length cDNA of BmHP14 is 2 508 bp long and has an open reading frame of 2 013 bp which encodes a protein of 670 amino acid residues with a predicted molecular mass of 71 kD,an theoretical point 5.09,and a predicted signal peptide of 17 amino acids at the N-terminus.Multiple sequence alignment showed that BmHP14 has a high sequence identity(57%) with Manduca sexta MsHP14,and they clustered into one group in phylogenetic tree.RT-PCR analysis revealed that BmHP14 was expressed in fat body,Malpighian tubule,testis,ovary,cuticle,haemocyte,head of day 3 silkworm larvae of the 5th instar,among which the expression in fat body was the highest.After day 3 silkworm larvae of the 5th instar were infected through injection with Bacillus bombyseptieus,Escherichia coli and Beauveria bassiana,Real-time PCR analysis indicated that BmHP14 was up-regulated in fat body.Western blotting showed that both the precursor and mature forms of BmHP14 protein existed in hemolymph of silkworm.These results demonstrated that BmHP14 is the key enzyme in the proPO cascade signal pathway of silkworm.