【作者】 丛海峰；

【导师】 司马杨虎； 徐世清；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2007， 硕士

【摘要】 家蚕是卵滞育昆虫，蚕业生产上通过低温冷藏和盐酸处理等方法解除蚕卵滞育。酯酶A4(Esterase A4，EA4)是一种能够感受蚕卵活化所需低温时间或酸刺激而发生蛋白质空间结构变化，获得瞬时ATPase活性的时间间隔测定酶(TIME)，被称作家蚕滞育生物钟蛋白质。本实验以家蚕C108品种为材料，根据本实验室完成的ea4基因cDNA的ORF序列(NCBI登陆号：DQ086424)，利用快速扩增cDNA末端法获得了其全长cDNA。测序结果表明，ea4基因全长cDNA 606 bp，ORF为519 bp，编码172个氨基酸。根据NCBI家蚕基因组数据，推断家蚕ea4基因至少含有4个外显子、3个内含子。由于家蚕全基因组数据存在缺口，通过拼接、修复GAP得到ea4基因从第2个外显子到第4个外显子全部序列，但第1个内含子序列未能确定。用核酸杂交技术以及半定量RT-PCR方法调查了ea4基因在家蚕5龄幼虫和蛹期不同组织的表达情况。随机引物地高辛(digoxygenin，DIG)标记ea4基因cDNA的核酸点杂交结果表明，在家蚕5龄幼虫和蛹期，ea4基因的表达不存在组织和性别特异性。采用Actin3基因做阳性对照，RT-PCR检测ea4基因的表达谱，结果在家蚕5龄幼虫和蛹期，ea4基因不存在发育时期的表达特异性，在丝腺、造血器官、中肠和脂肪体ea4基因的mRNA表达量较高，而在卵巢中表达量较低。证明了EA4蛋白质可能由家蚕造卵时期的幼虫至成虫期亲代非特异性合成后储存于卵。为了在基因表达水平进一步研究EA4蛋白质的生物测时机理，根据家蚕蛋白质EA4具有卵期瞬时ATPase活性(即生物钟活性)和整个卵期超氧化物歧化酶(Superoxide Dismutase，SOD)活性的特点，本文研究了家蚕以及野桑蚕ea4基因cDNA、EA4蛋白及SOD蛋白结构特点。比较家蚕和野桑蚕(NCBI：De154907)的ea4基因，两者的ORF同源性97％，蛋白同源性为99％。克隆野桑蚕的Cu／Zn-sod基因cDNA(NCBI登陆号：AM410997)后，推测的家蚕与野桑蚕SOD蛋白质氨基酸同源性为100％，家蚕EA4与SOD蛋白质的同源性为46％。家蚕EA4与Cu／Zn-SOD蛋白质有着相似的活性位点，包括金属配基氨基酸和二硫键，但N末端的相似性不高，这是EA4的特有结构，这部分是β-折叠重复空间结构，柔软可动，推测为EA4的计时功能的活性中心，是瞬时ATPase计时活性出现的关键结构，而EA4的钢性结构域可能是一直具有SOD活性的结构基础。从基因和蛋白质结构分析推测，家蚕EA4可能是起源于Cu／Zn-SOD家族的特异酯酶，家蚕EA4由幼虫至成虫期母体非特异性合成后储存于卵，其对环境低温、盐酸等活化刺激的时间间隔测定酶作用，依靠蛋白质空间结构变化测时，没有反馈环境刺激激活基因表达的调控方式。更多还原

【Abstract】 Domestic silkworm (Bombyx mori) is an egg diapause insect. In the production ofsilkworm, low-temperature chilling and hydrochloric acid treatment methods are used toterminate the diapause development. After above treatments, the spatial structure ofEsterase A4 (EA4) will be changed, and EA4 will get a transient burst AYPase activity.Thus it is named Time Interval Measuring Enzyme-Esterase A4 (TIME-EA4), acting as adispause duration clock in diapause eggs of the silkworm, Bombyx mori.Based on the ORF sequence of ea4 released by my laboratory (NCBI accessionnumber: DQ086424), we use rapid amplification cDNA end (RACE) to get the full lengthcDNA with the material of silkworm variety C108. The results show that the full lengthcDNA consists of 606 base pairs, and ORF is 519 base pairs coding 172 amino acids. Analigment of cDNA sequence with the whole genomic sequences of Bombyx mori onNational Center for Biotechnology Information (NCBI) revealed that there were 4 exonsand 3 introns. According to assembling and filling in GAPs of full genome data, thewhole sequence of ea4 gene was known except the first intron was uncertain.The expression of ea4 in different tissues of the fifth instar larva and pupa stage isresearched by nucleic acid hybridization and semi-quantitative RT-PCR method. WithDIG-High Prime labeled ea4 cDNA, the result of dot blotting shows that there was notissue and sex specificity of expression. With Actin3 gene as the positive control, theexpression profile of ea4 gene was detected by RT-PCR. Results showed that there wasno stage specificity in different tissues. Expression of ea4 is at higher level in silkgland,hemopoietic organs, midgut and fat body while the expression in ovary was lower. Wecome to conclusion that EA4 may be synthesized nonspeeificly by parents and thenstored in the eggs.Based on the transient burst of ATPase activity and SOD activity of EA4 during thetimer period, we researched the structure of ea4 gene, EA4 protein and SOD protein from Bombyx mori and Bombyx mandarina so that the bioclock mechanism of EA4 will befurther understood at gene expression level. The homology of ea4 ORF from Bombyxmori and Bombyx mandarina is 97%. And they share 99% at amino acid level. Thededuced amino acid sequence Cu/Zn-sod from Bombyx mandarina showes 100%identity with that of Bombyx mori. The homologies of EA4 with Bombyx mori andCu/Zn-SOD of Bombyx mori and Bombyx mandarina are 46%.The SOD active site including metal-binding amino acid ligands and a disulfidebond are completely identical in EA4 and SOD protein. The EA4 N-terminal region islow identities with SODs. The region is flexible because ofβ-sheet structure. The uniqueregion for EA4 is deduced to be the core domain to measure the transient burst of ATPaseactivity probably. The rigid core of EA4 has probably the SOD activity. According toanalyzing the structure of EA4 and SOD, EA4 of Bombyx mori is a specific easterase thatmay stem from SOD family. In a word, structure of EA4 will be changed and get atransient burst of ATPase activity treated by low-temperature chilling and hydrochloricacid. Then a time-dependent ATPase measures tne diapause duration of Bombyx daipauseeggs.更多还原