家蚕味觉电生理反应的个体差异

为探讨家蚕Bombyx mori人工饲料饲养发育不齐的生理原因,从同一蚕品种中挑选出对人工饲料摄食性不同的个体,用电生理学方法测定了下颚瘤状体味觉感器对4种代表性物质（蔗糖、肌醇、大豆粉提取物和柠檬酸）的电生理反应。结果表明,栓锥感器Ss-Ⅰ对蔗糖等促食物质的反应以及栓锥感器Ss-Ⅱ对大豆粉提取物等阻食物质的反应,均存在明显的个体差异。在临界浓度下,低摄食性个体的放电脉冲频率显著高于高摄食性个体。说明低摄食性蚕的味觉反应比高摄食性蚕敏感。     

家蚕化学感受蛋白CSP16的表达及结合特性分析

【目的】化学感受蛋白(chemosensory proteins,CSPs)在昆虫化学信号的感受识别和生长发育调节等生理过程具有重要作用。本研究旨在探索CSP16在家蚕Bombyx mori中的功能。【方法】利用RT-q PCR分析csp16在家蚕不同发育时期和不同组织的表达特征,用原核表达系统对家蚕CSP16进行表达纯化,并通过荧光结合实验检测该蛋白与不同配体化合物的结合特性。【结果】RT-q PCR结果表明,csp16在家蚕1-5龄幼虫中呈规律性表达,各龄期眠蚕中表达量最高,5龄第3天幼虫中主要表达于头、表皮、精巢和卵巢等。蜕皮激素(20E)处理后csp16在不同龄期幼虫和5龄幼虫不同组织中的表达量上调。纯化后CSP16的荧光结合实验表明,CSP16与醇类、酯类、醛类、酚类和苯环类等化合物的亲和力都较弱。【结论】csp16在家蚕不同龄期幼虫眠蚕中表达量最高,蜕皮激素使csp16在家蚕取食幼虫中的表达量出现上调,提示其可能参与家蚕幼虫的蜕皮过程。     

饲料中转基因成分对家蚕生长发育的影响

采用家蚕（Bombyx mori）H9品种作为实验动物,分别用转基因和非转基因大豆粉制作的人工饲料进行饲育。通过饲育试验,比较分析了不同处理后家蚕的龄期经过时间、全茧量、茧层量、茧层率、蛹体重、疏毛率、死亡率、每龄眠起体重等经济性状指标。同时采用PCR方法对转基因饲料饲育后家蚕组织中外源基因的表达情况进行了检测。结果显示,含转基因大豆粉的人工饲料对家蚕的生长发育并无显著影响,且不存在外源基因的污染。     

中国野蚕一种强抗病毒蛋白的基因分析和活性鉴定

以最近报道的家蚕抗病毒蛋白基因为线索,从中国野蚕（Bombyx mandarina Moore）中肠内克隆了抗家蚕BmNPV病毒的SP-2 cDNA（GenBank登录号：AY945210）,基因大小855bp,编码284个氨基酸的蛋白质,分子量29．6kD,基因组全长1376bp,包含5个外显子和4个内含子．该基因的表达仅限于中肠,具有组织特异性,在幼虫龄中表达水平较高,而在眠期和熟蚕没有表达．推导其氨基酸序列,发现其C端氨基酸序列与已报道的家蚕相应序列差别较大,有8个氨基酸完全不同．通过体外重组技术,由高效基因表达系统获得大量重组蛋白,发现该蛋白具有很强的抗家蚕BmNPV活性,与家蚕对应的抗病毒蛋白BmSP-2相比,其抗BmNPV活性高1．6倍．初步认为,该蛋白质C端序列差异可能是造成家蚕与野蚕抗病毒活性差别的主要原因．     

吡哆醛激酶基因在家蚕体内的时空表达特征

【目的】了解家蚕Bombyx mori维生素B₆关键代谢酶吡哆醛激酶（pyridoxal kinase, PLK）在家蚕不同组织间的表达差异。【方法】原核表达家蚕重组PLK, 获得目的蛋白制备多克隆抗体, 利用Western blot方法对PLK在家蚕不同发育时期、 5龄第3天幼虫不同组织及5龄不同日龄幼虫表皮、 头部和马氏管中的表达进行分析; 并采用实时荧光定量PCR的方法对PLK基因在家蚕不同组织中的mRNA转录水平进行比较。【结果】PLK在家蚕5龄幼虫的表达水平最高, 在5龄第3天幼虫各组织中的表达由高到低依次为： 精巢、 马氏管、 表皮、 中肠、 头部、 卵巢、 脂肪体、 丝腺; 5龄期不同日龄幼虫表皮组织的表达差异最大, 头部和马氏管中均为前期表达量稍高于后期。在转录水平方面, 精巢的表达量最高, 其次是马氏管和头部。【结论】PLK在家蚕不同发育时期及组织中的差异表达进一步证实PLK在家蚕VB6代谢中的生物学功能的重要性。     

家蚕人工饲料的研究:饲料理化因素对家蚕摄食和生长的影响

本文研究了人工饲料某些理化因素对蚕儿摄食和生长的影响,从而阐明家蚕生长发育过程中在摄食行为和营养上的特点和变化。 1.试验表明:绿原酸、桑色素、肌醇可增进蚁蚕摄食:β-谷固醇、没食子酸对保证小蚕正常发育甚为重要,绿原酸、桑色素、肌醇也有良好影响;相比之下,上述一些因子对5龄大蚕的作用较小。此外未看到β-谷固醇对蚁蚕的摄食促进效果。 2.大豆粉的醚溶性成份中,含有影响蚁蚕摄食的忌避物质。大豆粉经90%甲醇处理,可以除去某种水溶性因子,有利小蚕生长;对比之下,上列因素对大蚕的损害较小。比较了二种常见防腐剂对家蚕摄食、成长的影响:山梨酸的饲蚕成绩优于丙酸。 3.在本试验基本组成条件下,收蚁、小蚕、大蚕所用饲料的含水率分别以77%、73%、71%左右为宜。实验说明,蚕儿饲育效果还受饲料中具有成形作用组份的影响。 4.调查了饲料pH和蚕儿摄食、成长的关系:以某些适宜的有机酸(如柠檬酸、抗坏血酸)调节饲料的pH在5左右为好。 5.由于不同发育阶段的蚕儿对摄食、成长促进因子的感受性和对忌避因素的耐受性均不相同,从而提出,对蚕儿作全龄饲育,至少应分别设计为收蚁用、1—4龄小蚕用、5龄大蚕用三种饲料。此外,不同品种家蚕对人工饲料适应性的差别,与对饲料理化条件需求的不同也有一定联系。灵活运用上述结果,将有助于改良饲料组成,降低成本和广辟原料来源。     

家蚕几丁质去乙酰化酶BmCDA2的基因表达和免疫定位

几丁质的去乙酰化修饰与昆虫的发育变态密切相关,几丁质去乙酰化酶(chitin deacetylase,CDA)是这个过程中的关键酶。家蚕(Bombyxmori)是鳞翅目昆虫的代表性昆虫,目前对家蚕CDAs的研究较少。为了更好地揭示BmCDAs对家蚕变态发育的作用,本研究采用生物信息学分析、蛋白表达纯化以及免疫荧光定位等方法对表皮中高量表达的BmCDA2进行了研究。结果发现,BmCDA2有两种mRNA拼接形式BmCDA2a和BmCDA2b,分别在幼虫眠期和化蛹期表皮高量表达,两个基因均有几丁质去乙酰化酶催化结构域(catalyticdomain)、几丁质结合结构域(chitinbinding domain)和低密度脂蛋白受体结构域(low density lipoprotein receptor domain);Western blotting结果显示,该蛋白在表皮存在,荧光免疫定位发现BmCDA2蛋白随着幼虫新表皮的生成而逐渐增多,推测BmCDA2可能参与了幼虫新表皮的形成。该结果丰富了家蚕CDAs的生物学功能信息,也为其他昆虫CDA的研究提供一些有价值的参考。     

不同饲料饲育的家蚕幼虫肠道细菌的多样性分析

【目的】为研究饲料对不同家蚕Bombyx mori品种肠道微生物菌群的影响。【方法】以筛选到的家蚕广食性品种GS和普通品种1015为研究对象,收集从收蚁开始分别饲育桑叶(GS. m和C1015. m组)和人工饲料(GS. b组)至4龄盛时期的家蚕肠道样本,采用高通量测序的方法对其肠道微生物16S r DNA的V3-V4区进行测序分析,比较它们之间肠道微生物的差异。【结果】在门水平上,所测家蚕肠道样本的优势菌为厚壁菌门(Firmicutes)和变形杆菌门(Proteobacteria);在科水平上,所测样本主要优势菌为明串珠菌科(Leuconostocaceae)、乳酸杆菌科(Lactobacillaceae)、肠杆菌科(Enterobacteriaceae)等;在属水平上,所测样本主要的优势菌为魏斯氏属Weissella、乳酸菌属Lactobacillus、布赫纳氏菌属Buchnera、甲基杆菌属Methylobacterium、叶瘤菌属Phyllobacterium、肠球菌属Enterococcus和脆弱拟杆菌属Bacteroides等。家蚕品种GS经桑叶和人工饲料饲育后,甲基杆菌属Methylobacterium、布赫纳氏菌属Buchnera等菌属仅在桑叶饲育的GS肠道内出现,而魏斯氏菌Weissella、短芽孢杆菌属Brevibacillus等菌属只在人工饲料饲育的GS肠道内出现。同是桑叶饲育的家蚕品种GS和1015,其肠道内相同的优势菌有叶瘤菌属Phyllobacterium、脆弱拟杆菌属Bacteroides、不动细菌属Acinetobacter等。相较于广食性蚕品种GS的肠道菌群,肠球菌属Enterococcus、草螺菌属Herbaspirillum、丝硫菌属Thiothrix等菌属仅在普通蚕品种1015肠道中被检测到。GS. b组家蚕肠道细菌的物种多样性低于GS. m和C1015. m。GS. m肠道中丰度差异显著性最高的菌群为变形菌门(Proteobacteria),GS. b肠道中丰度差异显著性最高的菌群为杆菌纲(Bacilli)和乳杆菌目(Lactobacillales),而C1015. m肠道中丰度差异显著性最高的菌群为粪肠球菌属Enterococcus和肠球菌科(Enterococcaceae)。【结论】经桑叶饲育的不同蚕品种(GS和1015)的肠道微生物比人工饲料饲育的家蚕肠道微生物更趋于一致;经桑叶饲育的广食性家蚕肠道微生物物种多样性较高于经人工饲料饲育的广食性家蚕。     

家蚕磷酸吡哆醇氧化酶基因的表达谱分析

【目的】了解家蚕Bombyx mori维生素B6关键代谢酶磷酸吡哆醇氧化酶（pyridoxine- 5′-phosphate oxidase, PNPO）基因在家蚕不同发育阶段及5龄幼虫不同组织中的表达差异。【方法】将家蚕PNPO基因的重组表达质粒pET-22b(+)-PNPO转化入大肠杆菌Escherichia coli Rosetta中诱导表达, 纯化蛋白制备多克隆抗体。分别采用荧光定量PCR和Western blot方法对家蚕PNPO基因进行了转录水平和翻译水平的表达分析。【结果】在家蚕发育水平上, 5龄幼虫的PNPO翻译量为最高。PNPO基因在5龄幼虫各组织中的转录水平由高到低依次为精巢、 头、 中肠、 马氏管、 卵巢、 表皮、 脂肪体、 丝腺; 翻译量也以精巢为最高, 其次是头、 中肠和马氏管。【结论】明确了PNPO在家蚕各发育阶段及5龄幼虫各组织中的表达情况。     

家蚕幼虫中肠细菌群落多样性的PCR-DGGE和16S rDNA文库序列分析

采用基于16S rDNA 的变性梯度凝胶电泳（denaturing gradient gel electrophoresis, DGGE）和16S rDNA文库序列分析的手段,研究了重要经济昆虫家蚕Bombyx mori 2个品系——专食性品系C108和广食性品系SCN2幼虫中肠内的细菌群落多样性,同时还探讨了食料对家蚕中肠内细菌群落结构的影响。文库序列分析表明,PCR 扩增得到的16S rDNA基因代表了家蚕中肠内的41种细菌系统发育型（phylotype）,大多数属于Proteobacteria,其次是Lactobacillales。此外,还有少数属于Deinococcus-Thermus、Bacillales、Clostridiales和Actinobacteria,尚有5种系统发育型不能确定其所属类型。家蚕的这2个品系中,肠球菌属Enterococcus是其中肠细菌的优势菌群,栖热菌属Thermus是次优势菌群。优势菌肠球菌属的组成在品系和不同食料喂养条件下有着一定的变化,无桑饲料喂养条件下SCN2品系中肠内还出现了新的次优势菌葡萄球菌（Staphylococcus）。DGGE图谱显示家蚕低龄幼虫和高龄幼虫肠道细菌格局存在差异,推测可能与其发育期生理状态的差异有关。本研究结果提示家蚕肠道特殊菌群的出现可能与其特殊的食性有一定的关系,食料改变、生长受阻后肠道微生态平衡也发生变化。     

Differentially expressed genes in resistant and susceptible Bombyx mori strains infected with a densonucleosis virus

We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.     

Genome‐wide identification of chitin‐binding proteins and characterization of BmCBP1 in the silkworm,Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.     

Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori

Using microarray analyses, we identified carboxypeptidase A (MF-CPA), which was induced during pupal ecdysis in the wing discs of Bombyx mori. Here, we report the functional characterization of MF-CPA. MF-CPA has amino acid sequence similarities with the proteins in the carboxypeptidase A/B subfamily, from human to nematode. The MF-CPA gene is expressed during the molting periods in the epithelial tissues. MF-CPA is detected in the molting fluid, which fills the space between the old and new cuticle during molting. By Western blot analysis, we show that MF-CPA is secreted as a zymogen and processed in the molting fluid. Recombinant MF-CPA expressed in the insect cells has carboxypeptidase A activity. We propose that MF-CPA degrades the proteins from the old cuticle during the molting periods and contributes to recycling of the amino acids.     

饲料中维生素和无机盐对rBmNPV-Bm表达系统植酸酶基因表达活性的影响

【目的】为查明宿主的营养对重组家蚕杆状病毒-家蚕表达系统(rBmNPV-Bm)外源基因表达活性的影响,探寻提高家蚕Bombyx mori生物反应器产率的新途径。【方法】以家蚕BmNPV病毒为表达载体,以人工饲料饲养的5龄家蚕为宿主,以植酸酶基因为报告基因,探讨了家蚕人工饲料中维生素和无机盐对外源基因表达产物活性的影响。【结果】家蚕血淋巴中外源植酸酶的活性随着饲料中维生素和无机盐添加量的不同而发生显著变化。在本试验设区范围内,当维生素C的含量为饲料干物质量的1 %,B族维生素混合物为0.25 %,无机盐混合物为1 %,维生素B6为100 g饲料干物中含2 mg时,血淋巴中植酸酶活性达到最大值。【结论】通过改变家蚕饲料中微量营养成分的含量是提高rBmNPV-Bm表达系统外源基因表达活性的有效途径。     

A novel type of receptor cDNA from the prothoracic glands of the silkworm, Bombyx mori

A cDNA encoding a novel heptahelical receptor from the prothoracic glands of the silkworm, Bombyx mori was cloned and sequenced during screening of a prothoracicotropic hormone (PTTH) receptor. Orthologs of this receptor are found not only in insects, but also in the vertebrates. In B. mori, ubiquitous expression of the mRNA was observed in the larva. Also, a higher expression level in the prothoracic glands was observed before molting and metamorphosis and was impaired after pupal molting. But, further analysis is required to confirm whether this receptor cDNA encodes the PTTH receptor.     

CHITIN SYNTHASE B: A MIDGUT‐SPECIFIC GENE INDUCED BY INSECT HORMONES AND INVOLVED IN FOOD INTAKE IN Bombyx mori LARVAE

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third‐instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20‐hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry‐leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin‐synthesis‐dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae.     

Serial analysis of gene expression in the silkworm, Bombyx mori

The silkworm Bombyx mori is one of the most economically important insects and serves as a model for Lepidoptera insects. We used serial analysis of gene expression (SAGE) to derive profiles of expressed genes during the developmental life cycle of the silkworm and to create a reference for understanding silkworm metamorphosis. We generated four SAGE libraries, one from each of the four developmental stages of the silkworm. In total we obtained 257,964 SAGE tags, of which 39,485 were unique tags. Sorted by copy number, 14.1% of the unique tags were detected at a median to high level (five or more copies), 24.2% at lower levels (two to four copies), and 61.7% as single copies. Using a basic local alignment search tool on the EST database, 35% of the tags matched known silkworm expressed sequence tags. SAGE demonstrated that a number of the genes were up- or down-regulated during the four developmental phases of the egg, larva, pupa, and adult. Furthermore, we found that the generation of longer cDNA fragments from SAGE tags constituted the most efficient method of gene identification, which facilitated the analysis of a large number of unknown genes.     

Genetic mapping of a food preference gene in the silkworm, Bombyx mori, using restriction fragment length polymorphisms (RFLPs)

The domesticated silkworm, Bombyx mori, has strict food preferences and grows by feeding on mulberry leaves. However, "Sawa-J", an abnormal feeding habit strain selected from the genetic stock, feeds on an artificial diet without mulberry leaf powder. In this study, the food preference gene in Sawa-J was genetically identified using restriction fragment length polymorphisms (RFLPs) of a cDNA clone on each linkage group. Taking advantage of a lack of genetic recombination in females, reciprocal backcrossed F1 (BC1) progenies were independently prepared using a non-feeding strain, C108, as a mating partner of Sawa-J. Our results of linkage analysis and mapping proved that the feeding behavior is primarily controlled by a major recessive gene mapped at 20.2 cM on RFLP linkage group 9 (RFLG9), and clone e73 at a distance of 4.2 cM was found as the first linked molecular marker.