A chymotrypsin-like serine protease cDNA involved in food protein digestion in the common cutworm, Spodoptera litura: Cloning, characterization, developmental and induced expression patterns, and localization

A full-length cDNA (Slctlp2) encoding a chymotrypsin-like serine protease was cloned from Spodoptera litura. This cDNA encoded a putative serine protease with a predicted molecular mass of 30.6 kDa, which contained a serine protease catalytic motif GDSGGPL. Temporal and spatial expression of Slctlp2 mRNA and protein detected by Northern blotting, RT-PCR, qPCR and Western blotting analyses revealed that both Slctlp2 mRNA and protein were mainly present in the foregut and midgut of the 5th and 6th instar larvae during the feeding stages. In situ hybridization and immunohistochemistry confirmed that both Slctlp2 mRNA and protein were predominately present in the midgut. Expression of the gene was not induced by bacterial infection. Juvenile hormone III induced the gene expression, while 20-hydroxyecdysone had no impact on the expression. The expression of Slctlp2 mRNA and protein was down-regulated by starvation but up-regulated by re-feeding. The SlCTLP2 protein was detected in the lumen residues of the anterior, middle and posterior midgut and feces of the feeding 6th instar larvae, suggesting that it was secreted from the epithelium into the lumen of the gut. The results suggest that this Slctlp2 gene may be involved in digestive process of food proteins during the feeding stages of the larval development.     

CLONING AND CHARACTERIZATION OF A MIDGUT‐SPECIFIC FATTY ACID BINDING PROTEIN IN Spodoptera litura

A fatty acid binding protein (FABP) gene (Slfabp1) was cloned from the midgut of Spodoptera litura larvae. The gene consists of four exons and three introns and encodes a peptide of 134 amino acid residues with a predicted molecular mass of 14.7 kDa, which was confirmed by in vitro protein expression. Northern blot and Western blot analyses indicated that both of Slfabp1 mRNA and protein were highly and specifically expressed in the midgut during the fifth and sixth instar feeding larval stages. In situ hybridization and immunohistochemistry analyses confirmed the midgut‐specific localization of Slfabp1 mRNA and protein. The result of Western blot showed that expression of the protein was downregulated by starvation and upregulated by refeeding in sixth instar larvae. All of the results taken together suggest that the SlFABP1 plays important role(s) in FA uptake and transport in the midgut during the larval feeding stages of the insect.     

美国白蛾丝氨酸蛋白酶基因HcSP1的克隆、时空表达及对取食不同寄主植物的表达响应

【目的】为探究美国白蛾Hyphantria cunea在寄主转换过程中的消化生理机制奠定基础。【方法】通过筛选美国白蛾cDNA文库,克隆美国白蛾丝氨酸蛋白酶基因。荧光定量PCR检测该基因在美国白蛾不同发育阶段的表达特性;半定量RT-PCR和荧光定量PCR分别检测该基因在美国白蛾5龄幼虫体内不同组织中的分布及表达特性;荧光定量PCR检测取食不同寄主植物(美洲黑杨Populus deltoides,日本晚樱Cerasus serrulata var.lannesiana,山樱花Cerasus serrulata,喜树Camptotheca acuminata和法国梧桐Platanus orientalis)叶片后美国白蛾4龄幼虫中该基因的表达量。【结果】克隆获得美国白蛾丝氨酸蛋白酶基因HcSP1(GenBank登录号:MH663425),开放阅读框长882 bp,编码293个氨基酸,预测分子量为30.5 kD,理论等电点预测为9.86。编码蛋白N末端疏水区包含15个氨基酸组成的信号肽;具有丝氨酸蛋白酶的典型特征,即氨基酸序列中具有组氨酸(His)、天门冬氨酸(Asp)以及丝氨酸(Ser)残基组成的酶活性催化中心三元件;具有明显的胰蛋白酶前体的特征,即具有信号肽、激活肽以及胰蛋白酶N末端保守的起始氨基酸序列(IVGG)。NCBI BLAST比对结果表明美国白蛾HcSP1与其他鳞翅目昆虫丝氨酸蛋白酶的氨基酸序列一致性在50%～70%之间。荧光定量PCR结果显示,HcSP1在美国白蛾幼虫不同发育阶段的相对表达量呈现动态的变化,并随着幼虫虫龄的增长呈现上升趋势。半定量RT-PCR及荧光定量PCR结果显示,HcSP1在美国白蛾5龄幼虫头部、唾液腺、中肠、脂肪体、表皮、马氏管和血淋巴等组织中均有表达且在幼虫中肠中表达量极高。与取食其他寄主植物叶片相比,美国白蛾取食喜树叶片后HcSP1的相对表达量明显升高,并显著高于取食其他寄主植物。【结论】本研究克隆获得美国白蛾丝氨酸蛋白酶基因HcSP1,检测了其在美国白蛾不同发育阶段、不同组织以及取食不同寄主植物叶片后的表达量,为探究美国白蛾在寄主转换过程中消化生理的机制奠定基础,也为美国白蛾的防治提供新的思路。     

桃蛀螟CYP6AE76基因参与对Cry1Ab蛋白的解毒作用

【目的】明确桃蛀螟Conogethes punctiferalis幼虫取食Cry1Ab蛋白后体内CYP6AE76的过表达及对Cry1Ab蛋白有解毒作用。【方法】分析桃蛀螟CYP6AE76序列特征;利用RT-qPCR检测CYP6AE76在不同发育阶段(1-5龄幼虫)和4龄幼虫不同组织(头、中肠、血淋巴和脂肪体)以及4龄幼虫取食含有Cry1Ab蛋白(LC₅₀=1.08 ng/cm2)的人工饲料3 d存活的幼虫中肠和血淋巴中的表达量;利用RNAi饲喂法沉默桃蛀螟4 龄幼虫CYP6AE76后检测中肠中CYP6AE76的表达量,并统计120 h后幼虫体重并计算幼虫存活率;利用RNAi饲喂法沉默桃蛀螟初孵幼虫CYP6AE76后饲喂含1.08 ng/cm² Cry1Ab蛋白的饲料,7 d后统计幼虫体重并计算幼虫存活率。【结果】桃蛀螟CYP6AE76基因开放阅读框长1 572 bp,编码524个氨基酸,分子量约为60.34 kD,属于CYP6家族基因。发育表达谱结果表明, CYP6AE76基因在桃蛀螟整个幼虫阶段均有表达且在1龄幼虫期表达量最高,随着幼虫龄期增大而表达量降低;组织表达谱结果表明,CYP6AE76在4龄幼虫中肠中表达量最高。4龄幼虫取食含有Cry1Ab蛋白(1.08 ng/cm²)的人工饲料后,CYP6AE76在中肠和血淋巴中的表达量相比对照显著上调。通过RNAi沉默CYP6AE76后,桃蛀螟初孵幼虫再取食含有Cry1Ab蛋白的人工饲料后体重显著降低。【结论】CYP6AE76可能参与对桃蛀螟幼虫摄入的Cry1Ab蛋白的解毒。     

甜菜夜蛾肽聚糖识别蛋白基因SePGRP-SA的克隆及功能鉴定

【目的】本研究旨在阐明甜菜夜蛾Spodoptera exigua幼虫体内肽聚糖识别蛋白(peptidoglycan recognition protein, PGRP)在响应苏云金芽胞杆菌Bacillus thuringiensis(Bt)感染过程中的功能。【方法】利用PCR方法扩增甜菜夜蛾幼虫肽聚糖识别蛋白基因SePGRP-SA全长cDNA;采用qRT-PCR分析SePGRP-SA在甜菜夜蛾不同发育阶段(卵、1-5龄幼虫、预蛹和蛹)及4龄幼虫不同组织(中肠、马氏管、围食膜、脂肪体、血淋巴和表皮)中的表达。通过RNAi技术沉默SePGRP-SA基因72 h后,qRT-PCR检测SePGRP-SA沉默效率及甜菜夜蛾4龄幼虫中肠抗菌肽相关基因(Ceropin, Attacin和Defensin)和细菌载量的变化。RNAi沉默SePGRP-SA 24 h后,以苏云金芽胞杆菌菌株Bt-GS57饲喂甜菜夜蛾4龄幼虫0, 24, 48, 72, 96和120 h,计算幼虫校正死亡率;饲喂甜菜夜蛾4龄幼虫Bt-GS57后0, 24, 48和72 h,利用qRT-PCR检测中肠SePGRP-SA, Ceropin, Attacin和Defensin的相对表达量。【结果】克隆获得甜菜夜蛾SePGRP-SA全长DNA(GenBank登录号：MW265930),开放阅读框长576 bp,编码191个氨基酸,其编码蛋白的预测分子量为21.59 kD。序列分析结果表明,SePGRP-SA具有典型的PGRP和Ami2保守结构域,信号肽为19个氨基酸,为分泌型蛋白;系统进化分析发现,SePGRP-SA与斜纹夜蛾Spodoptera litura的SlPGRP亲缘关系最近,氨基酸序列一致性达91.1%。发育表达谱结果表明SePGRP-SA在甜菜夜蛾4和5龄幼虫、预蛹和蛹中高表达;组织表达谱结果表明,SePGRP-SA在4龄幼虫各组织中均表达,其中以血淋巴中表达量最高。与注射dsEGFP(对照)相比,注射dsSePGRP-SA的甜菜夜蛾4龄幼虫在72 h时中肠SePGRP-SA基因表达量下调了95.26%,Cecropin, Attacin和Defensin表达量显著下调,中肠细菌载量显著升高。注射dsEGFP和dsSePGRP-SA的甜菜夜蛾4龄幼虫饲喂Bt-GS57,72 h时幼虫校正死亡率分别为50.00%和73.33%,表明幼虫对Bt-GS57的敏感性明显增加。甜菜夜蛾4龄幼虫取食Bt-GS57后,中肠SePGRP-SA, Cecropin, Attacin和Defensin表达量在48 h均显著增加,72 h时降低。【结论】Bt侵染能够引起甜菜夜蛾SePGRP SA基因激活抗菌肽相关基因Cecropin, Attacin和Defensin的表达。     

Effects of rearing density on larval growth and activity of digestive enzymes in Lymantria dispar L. (Lepidoptera: Lymantriidae)

Density dependent responses of 4th, 5th and 6th instar gypsy moth larvae were studied at the level of larval mass, midgut loading and activities of three digestive enzymes (alpha-amylase, trypsin and leucine aminopeptidase). High density significantly reduced larval mass while midgut loading (expressed as relative midgut mass) did not change except in the 5th instar where it was increased at high density. Specific amylase and leucine aminopeptidase activities were not affected by crowding. Specific trypsin activity was on average higher in crowded than in isolated larvae. High density also affected the correlations between midgut protein content and activities of two proteolytic enzymes suggesting differences in regulatory mechanisms of insect digestion. The importance of these changes for survival under stressful conditions is discussed.     

棉铃虫叉头框蛋白A类似蛋白基因HarmFoxAl的克隆及表达谱分析

【目的】本研究旨在克隆并分析一种棉铃虫Helicoverpa armigera叉头框蛋白A (forkhead box protein A, FoxA)类似蛋白基因HarmFoxAl,探讨2-十三烷酮胁迫下棉铃虫中肠中HarmFoxAl的表达情况,为进一步明确棉铃虫FoxA的功能和参与棉铃虫生长发育的调控通路提供依据。【方法】从棉铃虫幼虫中肠中扩增得到HarmFoxAl的cDNA序列,并对其氨基酸序列和蛋白结构进行分析。将HarmFoxAl的ORF序列连接至pET32a载体并转化大肠杆菌Escherichia coli Transetta菌株,IPTG诱导后检测目的蛋白的表达形式,并利用镍柱亲和层析法纯化融合蛋白。通过qPCR检测棉铃虫不同发育阶段(1-6龄幼虫和预蛹),6龄幼虫不同组织(脂肪体、中肠、体壁和头部)以及10 mg/g 2-十三烷酮处理6龄幼虫不同时间后中肠中HarmFoxAl的表达谱。【结果】HarmFoxAl(GenBank登录号:XM021331806)的开放阅读框为669 bp,编码222个氨基酸,蛋白的相对分子质量和等电点分别为25.03 kD和6.34。氨基酸序列分析表明,HarmFoxAl单体蛋白无信号肽、跨膜区和二硫键,核心区域是由4个α螺旋和3个β折叠组成的球状结构。将重组的Transetta (pET32a-HarmFoxAl)菌株用0.5 mmol/L IPTG在25℃条件下诱导5 h,约45 kD的融合蛋白His-HarmFoxAl能以可溶的形式存在于重组菌中,这与预测的分子量(42.8 kD)相一致。发育阶段特异性表达谱表明,HarmFoxAl在棉铃虫1-3龄幼虫期、6龄幼虫期和预蛹期均有表达,且预蛹期的表达量最高。组织表达谱结果表明,该基因在6龄幼虫的脂肪体、中肠和体壁中表达,且脂肪体内的表达量最高,而在头部中不表达。10 mg/g 2-十三烷酮处理棉铃虫6龄幼虫后中肠中HarmFoxAl的表达量显著降低,但随着时间延长其表达量逐渐升高,处理48 h后表达量显著高于对照。【结论】棉铃虫HarmFoxAl在预蛹期和幼虫脂肪体中表达量最高,2-十三烷酮处理幼虫后HarmFoxAl的表达量急速降低后逐渐升高,推测其在棉铃虫变态发育和解毒代谢过程中发挥重要作用。     

Characterization of the molecular features and expression patterns of two serine proteases in Hermetia illucens (Diptera: Stratiomyidae) larvae

To investigate the molecular scavenging capabilities of the larvae of Hermetia illucens, two serine proteases (SPs) were cloned and characterized. Multiple sequence alignments and phylogenetic tree analysis of the deduced amino acid sequences of Hi-SP1 and Hi-SP2 were suggested that Hi-SP1 may be a chymotrypsin- and Hi-SP2 may be a trypsin-like protease. Hi-SP1 and Hi-SP2 3-D homology models revealed that a catalytic triad, three disulfide bonds, and a substrate-binding pocket were highly conserved, as would be expected of a SP. E. coli expressed Hi-SP1 and Hi-SP2 showed chymotrypsin or trypsin activities, respectively. Hi-SP2 mRNAs were consistently expressed during larval development. In contrast, the expression of Hi-SP1 mRNA fluctuated between feeding and molting stages and disappeared at the pupal stages. These expression pattern differences suggest that Hi-SP1 may be a larval specific chymotrypsin-like protease involved with food digestion, while Hi-SP2 may be a trypsin-like protease with diverse functions at different stages.     

Biochemical characterization of digestive proteases and carbohydrases of the carob moth,Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae)

Digestive proteinases and carbohydrases of Ectomyelois ceratoniae (Zeller) larvae were investigated using appropriate substrates and inhibitors. Midgut pH in larvae was determined to be slightly alkaline. Midgut extracts showed optimum activity for proteolysis of hemoglobin at pH 9–12. Midgut proteinases also hydrolyzed the synthetic substrates of trypsin, chymotrypsin, and elastase at pH 8–11. Maximum digestive α-amylase activity was also observed at pH 8–11. However, optimum activity for α- and β-glucosidase occurred at pH 5–8. Alpha- and β-galactosidases optimum activities occurred at pH 5 and pH 6, respectively. Inhibitors of serine proteases were effective on midgut serine proteases (trypsin and chymotrypsin proteases). Zymogram analyses revealed at least five bands of total proteolytic activity in the larval midgut. Protease-specific zymogram analyses revealed at least four, two, and one isozymes for trypsin-, chymotrypsin-, and elastase-like activities respectively. Two α-amylase isozymes were found in the midgut of fifth instar larvae and in the whole bodies of 1st through 5th instar larvae. Zymogram studies also revealed the presence of one and two bands of activity for β- and α-glucosidase, respectively. Recycling of α-amylase and proteases in the larval midgut was not complete. At least one isozyme of trypsin, chymotrypsin, elastase, and α-amylase were not recycled and were observed in the larval hindgut.     

Developmental and hormonal regulation of midgut remodeling in a lepidopteran insect, Heliothis virescens

Midgut tissue undergoes remodeling during metamorphosis in insects belonging to orders Lepidoptera and Diptera. We investigated the developmental and hormonal regulation of these remodeling events in lepidopteran insect, Heliothis virescens. In H. virescens, programmed cell death (PCD) of larval midgut cells as well as proliferation and differentiation of imaginal cells began at 108 h after ecdysis to the final larval instar (AEFL) and proceeded through the pupal stages. Expression patterns of pro- cell death factors (caspase-1 and ICE) and anti-cell death factor, Inhibitor of Apoptosis (IAP) were studied in midguts during last larval and pupal stages. IAP, Caspase-1 and ICE mRNAs showed peaks at 48 h AEFL, 96 h AEFL and in newly formed pupae, respectively. Immunohistochemical analysis substantiated high caspase-3 activity in midgut at 108 h AEFL. Application of methoprene, a juvenile hormone analog (JHA) blocked PCD by maintaining high levels of IAP, downregulating the expression of caspase-1, ICE and inhibiting an increase in caspase-3 protein levels in midgut tissue. Also, the differentiation of imaginal cells was impaired by methoprene treatment. These studies demonstrate that presence of JHA during final instar larvae affects both midgut remodeling and larval-pupal metamorphosis leading to larval/pupal deformities in lepidopteran insects, a mechanism that is different from that in mosquito, Ae. aegypti where JHA uncouples midgut remodeling from metamorphosis.     

Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression.

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.     

烟草甲clip丝氨酸蛋白酶基因的克隆及其对外源激素和免疫胁迫的表达响应(英文)

【目的】作为细胞外信号级联通路的重要组成部分,含有clip结构域的丝氨酸蛋白酶(clip-domain serine proteases, CLIPs)在昆虫发育和先天免疫过程中起着重要作用。本研究旨在克隆烟草甲Lasioderma serricorne CLIP基因,解析其在烟草甲不同发育阶段和幼虫不同组织中的表达模式,分析其在外源激素20-羟基蜕皮酮(20E)和免疫胁迫后的表达特征,为进一步研究其生理功能奠定基础。【方法】采用RT-PCR技术克隆获得烟草甲两个CLIPs基因(LsCLIP1和LsCLIP2)全长cDNA序列,并利用生物信息学软件预测其编码蛋白的结构和特征,利用MEGA 6.06构建昆虫CLIPs系统发育树;利用实时荧光定量PCR(quantitative real-time PCR, qPCR)研究这两个基因在不同发育阶段［低龄幼虫(卵孵化后24 h内)、高龄幼虫(4龄以上)、蛹(化蛹后48 h以上)、早期成虫(化蛹后24 h内)和晚期成虫(化蛹后7 d)］、5龄幼虫不同组织(表皮、脂肪体、肠道和剩余组织)中以及注射20E(120 ng/幼虫)和来源于大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus的肽聚糖(0.2 μL)后4龄幼虫中的表达模式。【结果】克隆获得烟草甲LsCLIP1和LsCLIP2基因的cDNA全序列,其开放阅读框长度均为1 194 bp,编码397个氨基酸。序列分析显示,其氨基酸序列各自具有一个clip结构域和胰蛋白酶结构域。系统发育分析表明,CLIP1和CLIP2都属于subfamily C CLIPs。qPCR结果表明,LsCLIP1和LsCLIP2基因在所检测的各发育阶段和幼虫各组织中均有表达,分别尤以蛹期和表皮中表达量最高;经20E和肽聚糖诱导后,烟草甲幼虫体内LsCLIP1和LsCLIP2基因的表达量明显提高。【结论】推测LsCLIP1和LsCLIP2可能参与了烟草甲的蜕皮发育和对免疫胁迫的应激响应。本研究将为后续研究昆虫CLIPs的分子调控提供参考。     

A cDNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in Helicoverpa armigera: molecular characterization and expression analysis associated with pupal diapause

The diazepam binding inhibitor (DBI) or the acyl-CoA-binding protein (ACBP) is a 9-10 kDa highly conserved multifunctional protein that plays important roles in GABA(A) receptor activity regulation, lipid absorption and steroidogenesis in various organisms. To study the functions of DBI/ACBP in insect development or diapause, we cloned the cDNA from Helicoverpa armigera (Har) utilizing rapid amplification of cDNA ends (RACE). By homology search, Har-DBI/ACBP is conserved with the DBI/ACBPs known from other insects. Northern blot analysis showed that DBI/ACBP gene expressed in nonneural and neural tissues. RT-PCR combined Southern blot analysis revealed that DBI/ACBP mRNA in the brain of nondiapause individual was much higher than that in the brain of diapausing insects. At early and middle stages of 6th instar larvae, the level of DBI/ACBP mRNA was higher in the midgut of diapause type than that in nondiapause type and low at late 6th instar larval stage and early pupal stage in both types. In the prothoracic gland (PG), DBI/ACBP expression appeared at a high level at middle and late stages of 6th larval instar in both nondiapause and diapause types, and declined after pupation. In vitro experiments revealed that DBI/ACBP mRNA in PG could be stimulated by synthetic H. armigera diapause hormone (Har-DH), suggesting that Har-DH may stimulate the PG to produce ecdysteroids by the DBI/ACBP signal pathway. By in vitro assay, we also found that FGIN-1-27, which has similar functions to DBI/ACBP in ecdysteroidogenesis, could induce PG ecdysteroidogenesis effectively, suggesting that DBI/ACBP regulates biosynthesis of ecdysteroids in PG. Thus, DBI/ACBP indeed plays a key role in metabolism and development in H. armigera.     

Ductin, a component of the V-ATPase, is developmentally regulated in Heliothis virescens midgut, and anti-ductin antibodies label lateral membranes

We previously cloned from Heliothis virescens a 16-kDa protein that is homologous to other ductin sequences. We also reported its immunolocalization with a specific affinity-purified anti-peptide antibody in the midgut and Malpighian tubule of feeding larvae, and concluded that the cloned proteolipid encodes the V-ATPase proton-transporting subunit c from the V₀ sector. We now present the immunolocalization of this protein in the midgut during the L₄-L₅ larval molt and early post-ecdysis into the fifth instar in H. virescens. The results show that the spatial expression of the 16-kDa protein is developmentally regulated. Labeling by anti-peptide antibody varies during the molt in the midgut goblet cell apical plasma membrane and the goblet cell apical valve. Epifluorescence and confocal microscopy revealed strong anti-ductin labeling in areas of cell-to-cell contact during the molt, and during early post-ecdysis into the fifth larval instar. The characteristic labeling pattern observed in areas of cell-to-cell contact is consistent with the claimed involvement of ductins in gap junctions. Conclusive evidence for the presence of the 16-kDa protein in areas of cell-to-cell contact in the midgut of feeding larvae is, however, lacking. V-ATPase regulation during the molt was also investigated by simultaneous immunohistochemistry with an anti-B subunit antiserum, a probe for the V₁ sector. Received: 2 April 1996 / Accepted: 14 November 1996     

A larval serum protein of the silkworm,Bombyx mori: cDNA sequence and developmental specificity of the transcript

The Bombyx mori larval serum protein (BmLSP) is a major component of larval hemolymph proteins until early in the last instar. The cDNA for BmLSP was cloned from a library constructed from fat body RNA of penultimate instar larvae, and the complete nucleotide sequence of the 909 base pair cDNA insert was determined. The deduced 262 amino acid polypeptide included a 16 amino acid residue signal peptide and a 15 amino acid sequence prosegment. A homology search showed that BmLSP has significant similarity with microvitellogenin of Manduca sexta and the 30K proteins of B. mori. Tissue distribution and developmental profile of BmLSP mRNA were analyzed by northern hybridization. BmLSP mRNA was abundant in fat body but not detected in midgut and silk gland. BmLSP mRNA was present during the feeding periods of the fourth and fifth instar larvae, but absent during the larval molt and after the onset of cocoon spinning.