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当前位置: 首页 > 文章 > 家蚕碱性丝氨酸蛋白酶基因Asp的克隆及在不同耐氟性家蚕组织中的表达差异 蚕业科学 2013,39 (3) 480-485
Position: Home > Articles > Molecular Cloning of Bombyx mori Alkaliphilic Serine Protease Gene and Differential Expression in Silkworm Tissues with Different Fluoride Endurance Ability Science of Sericulture 2013,39 (3) 480-485

家蚕碱性丝氨酸蛋白酶基因Asp的克隆及在不同耐氟性家蚕组织中的表达差异

作  者：
张华光;吴萍;徐安英;钱荷英;张月华
单  位：
江苏科技大学中国农业科学院蚕业研究所;中国农业科学院蚕业研究所
关键词：
家蚕;耐氟近等基因系;碱性丝氨酸蛋白酶;基因克隆;序列特征;组织表达;荧光定量PCR
摘  要：
在家蚕耐氟近等基因系SSH文库中发现一条差异表达的EST序列。以家蚕耐氟近等基因系群体的幼虫中肠cDNA为模板,利用RACE技术克隆了该基因的全长cDNA序列,将该基因命名为Asp(GenBank登录号:JX312360)。该基因cDNA全长868 bp,包括20 bp的5'-UTR和77 bp的3'-UTR,ORF为771 bp,编码256个氨基酸。该基因由4个外显子和3个内含子组成,编码蛋白质为亲水性蛋白,属于丝氨酸蛋白酶家族,在第17～18氨基酸残基处存在信号肽切割位点。荧光定量PCR检测该基因在家蚕耐氟近等基因系群体幼虫的中肠、脂肪体及马氏管中都有表达,以中肠的表达量最高,表达量因个体的耐氟性而存在差异:中肠中,敏感个体的表达量是耐氟个体的2.49倍;脂肪体中,敏感个体与耐氟个体之间的表达量差异不显著;马氏管中,敏感个体的表达量是耐氟个体的8.16倍。该基因在不同耐氟性家蚕品种5龄幼虫中肠的表达量存在极显著差异,在耐氟性品种的表达量是氟敏感品种的50倍。研究结果提示,该碱性丝氨酸蛋白酶基因可能与家蚕的耐氟性有一定的关联。
译  名：
Molecular Cloning of Bombyx mori Alkaliphilic Serine Protease Gene and Differential Expression in Silkworm Tissues with Different Fluoride Endurance Ability
作  者：
ZHANG Hua-Guang1 WU Ping2 XU An-Ying2 QIAN He-Ying2 ZHANG Yue-Hua2 SUN Ping-Jiang2 WANG Xiao-Qiang1(1Jiangsu University of Science and Technology,Zhenjiang Jiangsu 212003,China;2The Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang Jiangsu 212018,China)
关键词：
Bombyx mori;Near isogenic line of fluoride endurance;Alkaliphilic serine protease;Gene cloning;Sequence feature;Tissue expression;Fluorescent quantitative PCR
摘  要：
A differentially expressed EST fragment was discovered in the SSH library of Bombyx mori near isogenic line with fluoride endurance.Full length cDNA of this gene named as Asp(GenBank accession No.JX312360) was cloned by RACE method using midgut cDNA of Bombyx mori larvae from the population of near isogenic line with fluoride endurance.Full-length cDNA of this gene was 868 bp long,including 20 bp 5′-UTR and 77 bp 3′-UTR.Its ORF was 771 bp long and coded for 256 amino acids.This gene was composed of 4 exons and 3 introns.Its encoded protein was an alkaliphilic protein,belonging to the serine protease family.The protein had a cleavage site of signal peptide between the 17th and 18th amino acid residues.Real-time PCR revealed that this gene was expressed in all tested tissues including midgut,fat body and Malpighian tubule of Bombyx mori larvae from the population of near isogenic line with fluoride endurance.The expression level of Asp was the highest in midgut.Its expression level varied with the sensitivity of individuals to fluoride.In midgut,the expression level in sensitive individuals was 2.49 times to that in endurant individuals.In fat body,the expressional difference was not significant between sensitive and endurant individuals.In Malpighian tubule,the expression level in sensitive individuals was 8.16 times to that in endurant individuals.The expression level of Asp gene was obviously different in midgut of the 5th instar larva of various silkworm varieties with different fluoride endurance ability.The expression level in the fluoride resistant silkworm variety was 50 times to that in the fluoride sensitive variety.These results suggest that the alkaliphilic serine protease gene is associated with endurance to fluoride in Bombyx mori.

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