家蚕保幼激素结合蛋白Bmtol基因的克隆及功能分析

家蚕头部是一个神经中枢和感受的器官,其头部含有触角和感觉毛,感受外界的信号,并将外界信号传送到大脑进行反应。保幼激素主要是由咽侧体合成和分泌的,而保幼激素结合蛋白是保幼激素转运和发挥功能的载体,在昆虫体内具有极其重要的功能。文中通过Silk DB和NCBI数据库筛选并鉴定到一个新的具有保幼激素结合蛋白家族保守结构的蛋白Bm TOL,其编码基因编号为BGIBMGA003404(Gen Bank登录号:KY681053)。利用原核表达系统成功表达了该蛋白,通过Ni-NTA亲和层析的方法获得了Bm TOL的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平Bm TOL在头部都是高量表达,且Bmtol基因在起蚕时表达量较高,在5龄和蛹期表达量较低,而在化蛾后表达量又开始上调。免疫组化结果显示Bm TOL蛋白定位在头部的皮层、触角和脑中,推测其可能与头部信息传递有关,为家蚕的生长发育和行为调控提供重要的信息来源。     

Molecular cloning of pheromone biosynthesis activating neuropeptide in silkworm, Bombyx mori

Pheromone biosynthesis activating neuropeptide (PBAN) is a suboesophageal ganglion secretory polypeptide of insect, which activates the pheromone gland to produce sex pheromone biosynthesis in female silkworm, Bombyx mori. A Bombyx genomic library was screened by the method of plaque hybridization using the 32P-labeled BomDH cDNA as a probe. The genomic sequence encoding PBAN has been cloned and its structure is analyzed. The PBAN gene comprises two exons interspersed by a single intron 697 bp in length. Preceding the PBAN amino acid sequence is a 32-amino acid sequence containing two FXPRL amide peptides, which are α-SGNP (Ile-Ile-Phe-Thr-Pro-Lys-Leu) and β-SGNP (Ser-Val-Ala-Asn-Pro-Arg-Thr-His-Glu-Ser-Leu-Glu-Phe-Ile-Pro-Arg-Leu), which is followed by a Gly-Arg processing site. Immediately, after the PBAN amino acid sequence is a Gly-Arg processing site and a FXPRL amide peptide γ-SGNP (Thr-Met-Ser-Phe-Ser-Pro-Arg-Leu). It is suggested that besides PBAN, 7-, 8-, and 17-residue amidated peptides wer     

家蚕血淋巴体外黑化观察及相关黑色素合成催化酶基因

观察不同生长期家蚕幼虫血淋巴在体外的黑化速度和对大肠杆菌生长的影响结果显示,随食桑生长幼虫血淋巴的黑化速度逐渐变快,对大肠杆菌生长的抑制作用逐渐增强。RT-PCR实验显示,黑色素合成催化酶Bm Tan、Bm Po-1、Bm Yellow-f和Bm Ddc等的基因在家蚕5 L 3 d血淋巴中表达量高,Bm Black、Bm Yellow和Bm Pah等的基因也有明显表达。q PCR分析显示,黑化病蚕中Bmtan、Bmddc、Bmyellow、Bmebony和Bmblack,尤其Bmddc表达发生了显著上调。与对照相比,Ddc酶的抑制剂能显著抑制脂多糖对血淋巴的诱导黑化作用。用大肠杆菌注射家蚕幼虫,血淋巴中多巴和多巴胺的含量明显上升。这些表明家蚕幼虫血淋巴黑化与防御免疫有关,Bmddc很可能在幼虫血淋巴的免疫黑化中发挥作用。     

Evidences for a stage-specific juvenile hormone binding protein in the hemolymph of the silkworm,Bombyx mori L.: Identification and characterization by photoaffinity labeling and immunological analyses

Two molecular forms of juvenile hormone binding proteins were identified in the larval hemolymph of Bombyx mori by photoaffinity labeling. One form having an Mr of 33 kDa was present constantly in the hemolymph of the third to the fifth instar larvae while the other form having an Mr of 35 kDa was detected in the hemolymph until in the early fifth instar larvae but not in the prewandering larvae and prepupae. A 33 kDa binding protein was purified by hydrophobic interaction chromatography, gel filtration, and native PAGE. Antiserum against 33 kDa binding protein cross-reacted with 35 kDa binding protein on Western blots, suggesting that these binding proteins shared the same epitopes. From the results of saturation binding assays, it was inferred that 33 and 35 kDa binding proteins had a similar binding affinity for JH 1. It was revealed that one of these binding proteins, 35 kDa binding protein, was produced in the fat body in a stage-specific manner: fat body of the early fifth instar larvae synthesized both 33 and 35 kDa binding proteins while that of prewandering larvae synthesized only 33 kDa binding protein. © 1996 Wiley-Liss, Inc.     

外源激素处理后家蚕吡哆醛激酶和磷酸吡哆醇氧化酶转录水平的变化

【目的】研究家蚕 Bombyx mori 经蜕皮激素(20-hydroxyecdysone, 20-E)和保幼激素类似物(juvenile hormone analogue, JHA)处理后引起吡哆醛激酶(pyridoxal kinase, PLK)和磷酸吡哆醇氧化酶(pyridoxine-5′-phosphate oxidase, PNPO)的转录水平变化,为进一步研究激素对蚕体营养代谢等工作奠定基础。【方法】以20-E和JHA分别喂食不同发育时期(5龄第1, 3和5天)的家蚕幼虫,以喂食蒸馏水的家蚕为对照,采用实时荧光定量PCR(real-time quantitative PCR)方法在处理后24 和48 h对各组幼虫后部丝腺中PLP合成酶PLK和PNPO的转录水平进行分析。【结果】5龄第1天幼虫经20-E处理24和48 h后,PLK和PNPO的转录水平出现上调且与对照的差异达到极显著 (P＜0.01);5龄第3天幼虫经20-E处理,PLK的转录水平在48 h出现下调且与对照的差异达到显著(P＜0.05),PNPO的转录水平在24 和48 h均出现上调且与对照的差异达到极显著 (P＜0.01);5龄第5天幼虫经20-E处理后PLK和PNPO的转录水平无变化。5龄第1天幼虫经JHA处理后PLK和PNPO的转录水平未受到影响;5龄第3天幼虫经JHA处理后,PLK的转录水平在48 h出现显著下调且与对照的差异达到显著(P＜0.05),PNPO的转录水平在24和48 h后均出现显著下调且与对照的差异达到极显著(P＜0.05);5龄第5天幼虫经JHA处理24和48 h后,PLK和PNPO的转录水平出现下调且与对照的差异达到极显著 (P＜0.01)。【结论】20-E和JHA显著影响家蚕5龄幼虫PLK和PNPO的转录水平,20-E提高5龄前期家蚕PLK和PNPO的转录水平,JHA降低5龄后期它们的转录水平,为深入研究激素对VB₆的调控奠定基础。     

Identification and characterization of the diuretic hormone 31 receptor in the silkworm Bombyx mori

The receptor for diuretic hormone 31 (DH31R) was identified in the silkworm Bombyx mori. A heterologous expression system revealed that an orphan G-protein coupled receptor, BNGR-B1, responded to DH31 and upregulated the intracellular cAMP level. DH31R (BNGR-B1) was predominantly expressed in the anterior silk gland, midgut, and ovary, whereas DH31 was predominantly expressed in the central nervous system and midgut.     

家蚕滞育激素-性信息素合成激活肽基因表达的调节

滞育激素和性信息素合成激活肽是两个重要的昆虫神经肽,这两个神经肽由一个基因编码．利用分子杂交和ＲＴ－ＰＣＲ技术,确定了滞育激素－性信息素合成激活肽基因表达的调节不属于转录后的调节,推定为翻译后形成一个大的前体多肽再剪接为几个成熟的神经肽分子．     

Analysis of precocious metamorphosis induced by application of an imidazole derivative to early third instar larvae of the silkworm,Bombyx mori

When an imidazole derivative (KK-42) was applied to day 1 third instar larvae of the silkworm, Bombyx mori, 100% underwent precocious metamorphosis at the end of the fourth instar. Thus, the fourth instar becomes the last instar in these KK-42–treated larvae. The endocrine systems underlying the precocious metamorphosis were analyzed in the present study. Hydroprene application during the prolonged third instar after KK-42 treatment can prevent precocious metamorphosis, and the results showed dose-dependent and stage-specific effects. From analysis of the developmental changes in ecdysteroid levels in both KK-42–treated larvae and KK-42– and hydroprene-treated larvae, we conclude that changes in JH levels during the third larval instar can modify the secretion pattern of prothoracic glands and that during the next larval instar, very low ecdysteroid levels during the early stages of the presumptive last (fourth) larval instar are directly related to precocious metamorphosis. Arch. Insect Biochem. Physiol. 36:349–361, 1997. © 1997 Wiley-Liss, Inc.     

Juvenile hormone acid methyl transferase is a key regulatory enzyme for juvenile hormone synthesis in the Eri silkworm, Samia cynthica ricini

During the period of adult emergence in the Eri silkworm, Samia cynthia ricini, the corpora allata (CA) are apparently reactivated in females, but not males. This creates a significant sexual dimorphism in juvenile hormone (JH) synthesis by CA. To determine the underlying molecular mechanisms in this process, we cloned cDNAs of two enzymes involved in the JH synthesis pathway: 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and juvenile hormone acid methyl transferase (JHAMT). Both Samcri-HMGR and -JHAMT mRNAs were detected in CA almost exclusively. However, their expression patterns were different from each other. During the period of adult emergence, Samcri-HMGR was expressed in CA at a constantly high level suggesting it plays little role for the regulation of JH synthesis. In contrast, the patterns of both Samcri-JHAMT mRNA level and enzyme activity were closely correlated with the patterns of JH synthesis, CA reactivation, and sexual dimorphism of JH synthesis. In addition, JHAMT mRNA levels were paralleled JH synthesis in the fifth-instar larvae of S. cynthia ricini and the pharate adults of the silkworm Bombyx mori. We infer from these results that JHAMT is a key regulatory enzyme for JH synthesis in the Eri silkworm.     

转录因子ZnF-706在鳞翅目昆虫中的进化格局及在家蚕中的功能

【目的】探讨家蚕Bombyx mori的潜在驯化基因——转录因子ZnF-706在鳞翅目(Lepidoptera)昆虫进化过程及家蚕驯化过程中的分子进化格局;并基于CRISPR/Cas9家蚕基因组编辑平台,探讨ZnF-706基因在家蚕中的功能。【方法】首先分析了家蚕ZnF-706序列特征,并利用已发表芯片数据调研该基因在家蚕幼虫组织中的表达格局;利用Phylogenetic Analysis byMaximum Likelihood (PAML)分支检验方法,分析该基因在鳞翅目不同类群中的分子进化格局。基于已发表的家蚕-野桑蚕Bombyx mandarina群体基因组多态性数据,对ZnF-706进行基因区域人工选择信号分析;对ZnF-706基因上游2 kb的调控区域进行单核苷酸多态性位点频率检测,发掘在家蚕群体中固定下来的突变位点;针对突变位点所在区域进行转录因子结合活性预测。利用CRISPR/Cas9基因编辑技术敲除ZnF-706基因,获得纯和突变体;以野生型家蚕为对照,检测突变体的茧重及蛹重变化。【结果】家蚕ZnF-706的编码蛋白具有典型的锌指蛋白结构域。ZnF-706在家蚕5龄第3天幼虫各组织中广泛表达,尤其表皮、脂肪体和生殖腺中有很高的表达量;该基因在鳞翅目、蚕蛾总科(Bombycoidea)及天蚕Antherea yamamai 3个分支中均呈现快速进化信号,在家蚕中有强烈的人工选择信号。该基因所在基因组区域的家蚕-野桑蚕种群分歧度参数Fst明显升高,家蚕群体中的群体多样性π明显降低,表明它位于一个选择扫荡区域内;该基因在家蚕-野桑蚕中的9个SNP位点存在于上游调控区,并位于转录因子结合活性区域内。该基因的纯合家蚕突变体ΔZn F-706生存力减弱,并且茧重以及蛹重与野生型家蚕相比都显著降低。但与黑腹果蝇Drosophila melanogaster中不同的是,家蚕中该基因的突变并不致死。【结论】ZnF-706可能在鳞翅目尤其是泌丝昆虫中进化,并在家蚕驯化过程中受到选择压力,提示其对于特征性状茧丝的变异可能发挥作用。该基因可能通过对丝蛋白基因的直接调控,或通过影响家蚕的生长发育而间接地影响茧丝性状。本研究不仅为探究家养动物人工选择机制提供了来自昆虫类材料的独有证据,也为后续深入开展家蚕重要经济性状的转录调控研究提供线索。     

家蚕Fox转录因子在精巢中的表达特征及BmFoxL2在精巢发育中的功能初探

Fox家族蛋白广泛存在于植物以外的物种中,是一类与发育相关的重要转录因子.但是不同Fox基因在鳞翅目昆虫家蚕Bombyx mori精巢中的表达特征,以及BmFoxL2亚家族蛋白在精巢发育中的功能未知.本文检测了家蚕12个BmFox基因在家蚕精巢中的表达量,发现BmFoxL2-2的表达量最高,BmFoxL2-1次之.Bm...     

用于细胞水平家蚕转录调控元件研究的简单基础启动子的构建

【目的】本研究致力于构建一种能够在家蚕Bombyx mori细胞水平稳定表达的简单基础启动子,从而更准确地反映单一转录调控元件对基因启动子活性的影响,为研究家蚕乃至其他昆虫的基因转录调控奠定基础。【方法】本研究在本课题组已报道的能在家蚕细胞中稳定表达且基本不含上游转录调控元件的BmVgP78M启动子的基础上,通过PCR技术在其上游添加一定长度的间隔序列和能够应答20-羟基蜕皮激素(20E)且增强启动子活性的BrC-Z2转录因子结合基序(BrC-Z2 element, BrC-Z2E);通过基因克隆技术构建细胞转染载体;通过细胞转染技术和双荧光素酶报告基因系统检测启动子活性的变化。【结果】通过在BmVgP78M启动子上游添加28 bp间隔序列,成功构建了一个简单基础启动子,命名为VgP78ML,并证明其为可用于研究目标转录调控元件的简单基础启动子。经实验验证表明,该简单基础启动子不仅可以在家蚕细胞中稳定表达,且其本身活性不受20E及转录因子BrC-Z2的影响;当该启动子上游连接BrC-Z2E时,可以显著地应答20E及BrC-Z2转录因子,从而调控报告基因的表达。【结论】VgP78ML能够作为简单基础启动子应用于细胞水平对家蚕基因转录调控进行研究。同时,其构建方法也为其他物种构建研究转录调控的简单基础启动子提供了参考。     

Functional analysis of four Gloverin-like genes in the silkworm, Bombyx mori

To identify genes involved in the innate immunity of the silkworm Bombyx mori, we constructed a cDNA library from the fat body of Escherichia coli-challenged B. mori larvae. Based on the expressed sequence tag (EST) data and whole genome shotgun sequence analysis, we found four Gloverin-like genes, BmGlov1-4, in the Bombyx genome. Northern blot and RT-PCR analysis showed that BmGlov1-4 were induced in the larval fat body after an immune challenge by the injection of E. coli; however, less induction was observed after the injection of a yeast Candida albicans. In silico sequence analysis revealed the presence of a motif homologous to NF-kappaB binding site in the upstream region of each BmGlov gene. Moreover, we expressed recombinant BmGlov1-4 proteins using the baculovirus expression system, and found that all the recombinant BmGlov1-4 significantly inhibited the growth of E. coli.     

Expression of juvenile hormone acid O‐methyltransferase and juvenile hormone synthesis in Blattella germanica

Juvenile hormone (JH), a sesquiterpenoid synthetized by the insect corpora allata (CA), plays critical roles in metamorphosis and reproduction. Penultimate or last step of JH synthesis is catalyzed by juvenile hormone acid O‐methyltransferase (JHAMT). Here we report the cloning and expression analysis of the JHAMT orthologue in the cockroach, Blattella germanica (L.) (BgJHAMT). BgJHAMT is mainly expressed in CA, with only expression traces in ovary. Three different isoforms, differing in the 3′‐UTR sequence, were identified. Isoform A shows between 35 and 65 times higher expression than B and C in CA from penultimate nymphal instar and adult females. RNAi‐triggered knock down of BgJHAMT produces a dramatic reduction of JH synthesis, concomitant with a decrease of fat body vitellogenin expression and basal follicle length. BgJHAMT mRNA levels in CA of females along the gonadotrophic cycle parallel, with a slight advancement, JH synthesis profile. BgJHAMT mRNA levels were reduced in starved females and in females in which we reduced nutritional signaling by knocking down insulin receptor and target of rapamycin (TOR). Results show that conditions that modify JH synthesis in adult B. germanica females show parallel changes of BgJHAMT mRNA levels and that the JH‐specific branch of the JH synthesis pathway is regulated in the same way as the mevalonate branch. Furthermore, we demonstrate that nutrition and its signaling through the insulin receptor and TOR pathways are essential for activating BgJHAMT expression, which suggests that this enzyme can be a checkpoint for the regulation of JH production in relation to nutritional status.