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Sima YH Yao JM Hou YS Wang L Zhao LC 《Archives of insect biochemistry and physiology》2011,77(2):72-80
For diapause eggs of the silkworm, Bombyx mori, diapause initiation is prevented with hydrochloric acid (HCl) at around 20 h post-oviposition while diapause status is terminated with chilling around 5°C. To investigate whether hydrogen peroxide (H(2)O(2)) and catalase expression are involved in diapause initiation and termination, the concentration of H(2)O(2), relatively higher levels of catalase mRNA and activity of catalase were compared between (1) 20-h-old diapause eggs and the HCl-treated diapause eggs, and (2) 10-day-old diapause eggs and the 5°C-chilled diapause eggs. Compared to diapause eggs, the HCl-treated eggs had significantly higher H(2)O(2) concentrations (up from approximately 1-3 μmol/g fresh mass to 5-8 μmol/g fresh mass), higher relative level of catalase mRNA (up from 0 to 35.2%) and higher catalase activity (up from 2.51 units/mg protein to 4.97 units/mg protein) at 96 h post-treatment. On the other hand, the 5°C chilling resulted in significant increases of H(2)O(2) concentration (up from 0.79 μmol/g fresh mass to 5.57 μmol/g fresh mass), relative level of catalase mRNA (up from 0 to 71.4%) and catalase activity (up from 0.88 units/mg protein to 3.42 units/mg protein) within 120 days. The results obtained in this work suggest that variations of H(2)O(2) and catalase expression in Bombyx eggs are involved in diapause initiation and termination. 相似文献
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家蚕Bombyx mori由受精卵到完成胚胎发育孵化的过程中, 细胞进行大量的分裂和分化, 然而滞育性卵的胚胎细胞分化至G2期便停滞在此阶段。为了探索这一发育阶段细胞内的分子调控, 本研究以人Homo sapiens的细胞周期蛋白基因cyclin L1为模板, 成功克隆了家蚕同源基因BmCcnl1（GenBank登录号: FJ889988）。BmCcnl1基因开放阅读框（open reading frame, ORF）全长1 254 bp, 编码417个氨基酸。利用Protean软件分析得出BmCcnl1蛋白预测分子量为49 kDa, 等电点为9.84。利用DNA重组技术构建了BmCcnl1基因的重组表达载体pET-21d-BmCcnl1, 对其进行原核表达, 其表达的蛋白以包涵体形式存在。利用RT-PCR技术分析了BmCcnl1基因在胚胎发育过程中的转录水平, BmCcnl1基因在非滞育性卵的胚胎发育阶段基本保持相对稳定的转录表达, 而滞育性卵从蛾体产下经过72 h后已经检测不到BmCcnl1基因的转录。结果提示, BmCcnl1基因与胚胎期滞育及非滞育性卵的发育调控相关。对该基因的克隆和表达分析为今后研究家蚕胚胎发育及细胞周期调控奠定了基础。 相似文献
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早期胚胎显微注射是目前获得转基因家蚕Bombyx mori的主要途径。显微注射操作对蚕卵的损伤导致注射后的蚕卵孵化率降低, 是家蚕转基因工作的主要障碍之一。本研究对不同卵龄的蚕卵进行了开孔或注射实验, 并对产后5 h的蚕卵上背侧、 腹侧、 前极、 后极和中央等5个不同的位置进行了开孔实验, 调查了卵孵化率和体形异常蚕的产生情况。结果表明： 较早卵龄期的注射或从蚕卵背侧的注射可以获得高的孵化率。腹侧注射产生大量的体形异常蚕而背侧注射的蚕完全正常。通过调整注射时期和注射位置避开上述影响可以减少死卵和畸形蚕, 提高孵化率。本研究为改进家蚕转基因操作技术提供了有效的参考。 相似文献
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Abstract. An in vitro culture method is described in which embryonic development in Bombyx mori is traced at various temperatures and treatments. The results show that the induction, intensification and termination of diapause are distinct processes. Prediapause embryos, explanted from 40-h-old diapause-destined eggs and cultured in Grace's medium, continue to develop to the appendage-formation stage without arrest, which indicates that the isolated embryos have not entered diapause, whereas the development of embryos from diapausing eggs (15 days after being laid) is significantly slower. The rate of development of embryos dissected from diapause eggs increases during chilling (5 °C) and incubation (at 25 °C) gradually during chilling and dramatically at 25 °C. The in vitro experiments also reveal that sorbitol directly inhibits the development of embryos explanted from diapausing eggs but has no affect on the development of embryos from prediapause eggs. Neither alanine nor diapause hormone prevent isolated embryos from developing. 相似文献
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不同催青方式对二化性家蚕过氧化氢酶基因表达的影响 总被引：3，自引：0，他引：3
25℃明催青和15℃暗催青分别诱导二化性家蚕Bombyx mori 产滞育性卵和非滞育性卵。此前我们的研究表明, 上述催青处理的二化性家蚕H2O2水平存在显著差异。过氧化氢酶（catalase, CAT）是昆虫清除H2O2的关键酶。为了进一步明确家蚕滞育过程中H2O2代谢的调控机制, 用RT-PCR测定了上述两种催青处理对二化性家蚕CAT基因表达的影响。结果表明：25℃明催青显著提高了滞育诱导和决定阶段的CAT mRNA 水平和CAT活性。滞育性卵的CAT mRNA水平在产后24 h形成峰值, 在72 h后消失; CAT活性在96 h前上升, 120 h后保持于低水平。非滞育性卵的CAT mRNA水平和CAT活性都随着胚胎发育而上升。可见, 25℃明催青诱导二化性家蚕子代滞育可能是通过影响CAT基因表达来调节H2O2水平。 相似文献
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To explore whether glutathione regulates diapause determination and termina tion in the bivoltine silkworm Bombyx mori, we monitored the changes in glutathione redox cycle in the ovary of both diapanse and nondiapauseegg producers, as well as those in dia pause eggs incubated at different temperatures. The activity ofthioredoxin reductase （TrxR） was detected in ovaries but not in eggs, while neither ovaries nor eggs showed activity of glutathione peroxidase. A lower reduced glutathione/oxidized glutathione （GSH/GSSG） ratio was observed in the ovary of diapauseegg producers, due to weaker reduction of oxidized glutathione （GSSG） to the reduced glutathione （GSH） catalyzed by glutathione reductase （GR） and TrxR. This indicates an oxidative shift in the glutathione redox cy cle during diapause determination. Compared with the 25℃treated diapause eggs, the 5℃treated diapause eggs showed lower GSH/GSSG ratio, a result of stronger oxidation of GSH catalyzed by thioredoxin peroxidase and weaker reduction of GSSG catalyzed by GR. Our study demonstrated the important regulatory role of glutathione in diapause determination and termination of the bivoltine silkworm. 相似文献
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R. V. SESHACHALAM M. V. V. SUBRAMANYAM R. V. KRISHNAMOORTHY† 《Physiological Entomology》1992,17(3):277-280
Abstract. Silkworm eggs of diapause nature were chilled or treated with hydrochloric acid. Glutamine synthetase activity in such treated eggs was present soon after the treatment, whereas in non-diapause eggs it was not detectable until 24 h after the start of development. During larval life, the glutamine synthetase was found to be absent in midgut tissue. Topical application of a JH analogue resulted in extened larval duration and it reduced glutamine synthetase activity initially, but in the latter part of development the activity was higher. 相似文献
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Sasibhushan Sirigineedi Esvaran Vijayagowri Geetha N. Murthy Guruprasada Rao Kangayam M. Ponnuvel 《Insect Science》2014,21(6):677-686
A comparison of the cDNA sequences （1 056 bp） of Bombyx mori DnaJ 5 homolog with B. mori genome revealed that unlike in other Hsps, it has an intron of 234 bp. The DnaJ 5 homolog contains 351 amino acids, of which 70 contain the conserved DnaJ domain at the N-terminal end. This homolog orB. mori has all desirable functional domains similar to other insects, and the 13 different DnaJ homologs identified in B. mori genome were distributed on different chromosomes. The expressed sequence tag database analysis of Hsp40 gene expression revealed higher expression in wing disc followed by diapause-induced eggs. Microarray analysis revealed higher expression of DnaJ 5 homolog at 18th h after oviposition in diapause-induced eggs. Further validation of DnaJ 5 expression through qPCR in diapause-induced and nondiapause eggs at different time intervals revealed higher expression in diapause eggs at 18 and 24 h after oviposition, which coincided with the expression of Hsp70 as the Hsp 40 is its co-chaperone. This study thus provides an outline of the genome organization of lisp40 gene, and its role in egg diapause induction in B. mori. 相似文献
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家蚕胚胎发育中过氧化氢的代谢(英文) 总被引：2，自引：0，他引：2
过氧化氢（Ｈ_２Ｏ_２）是生物体内主要的活性氧来源之一。在超氧化物歧化酶（ＳＯＤ）、过氧化氢酶（ＣＡＴ）等的催化作用下,Ｈ_２Ｏ_２。被降解,释放出活性氧。所以,生物个体发育过程中体内Ｈ_２Ｏ_２、ＳＯＤ和ＣＡＴ含量的变化反映着Ｈ_２Ｏ_２的代谢水平。另外,家蚕是蚕卵滞育昆虫,实验设计考虑到了滞育前后可能会有的差别。取产后１０分钟内的卵为供试材料。采用即时浸酸法解除卵滞育。采用比色法和氧电极法测定并比较家蚕胚胎滞育形成与解除过程中过氧化氢的代谢。结果表明：（１）受精初期（０～４ｈ）,Ｈ_２Ｏ_２含量在２．５ｈ时达到峰值（Ｆｉｇ．１）,相应地ＳＯＤ活性处于较高水平,而ＣＡＴ活性处于最低水平（Ｆｉｇ．２）;（２）胚胎发育过程中（即时浸酸解除滞育）,Ｈ_２Ｏ_２含量除１６８～２１６ｈ处于低水平外均显著高于滞育卵（Ｆｉｇ．３）,ＳＯＤ活性分别在７２ｈ、１６８ｈ,形成小大两峰,后期显著高于滞育卵（Ｆｉｇ．４）,而ＣＡＴ活性７２－１９２ｈ保持平稳,随后急剧上升,前期显著低于滞育卵,后期相反（Ｆｉｇ．５）;（３）滞育形成过程中Ｈ_２Ｏ_２水平变化平缓（Ｆｉｇ．６）,ＳＯＤ活性前期剧烈变动,但后期保持平稳（Ｆｉｇ．７）,ＣＡＴ活性逐步升 相似文献
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Ai‐Chun Zhao Ding‐Pei Long San‐Yuan Ma Long‐Xia Xu Mei‐Rong Zhang Fang‐Yin Dai Qing‐You Xia Cheng Lu Zhong‐Huai Xiang 《Insect Science》2012,19(2):172-182
Abstract To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time‐consuming to maintain non‐diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HCl treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incubating developing mother eggs from Chinese lineage diapause silkworm strains at 15°C (15°C‐IME), we were able to prevent the diapause of their daughter eggs; a similar strategy (15°C‐IMES) for the germline transformation of the same strains was that the mother eggs were incubated at 15°C, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non‐diapause eggs. By combining temperature and light controls, the improved 15°C‐IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15°C‐IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling. 相似文献
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家蚕滞育卵与非滞育卵中几种关键酶活性的比较 总被引：2，自引：0，他引：2
家蚕Bombyx mori是卵滞育的昆虫, 在滞育期间无形态变化, 也不存在器官发育和组织分化, 然而其生理代谢过程仍在进行。为进一步研究家蚕滞育的机制, 本研究测定了家蚕滞育卵、 即时浸酸处理的滞育卵及非滞育卵在胚胎发育过程中的超氧化物歧化酶(superoxide dismutase, SOD, EC 1.15.1.1)、 过氧化氢酶(catalase, CAT, EC 1.11.1.6)、 丙酮酸激酶(pyruvate kinase, PK, EC 2.7.1.40)、 乙酰胆碱酯酶(acetylcholine esterase, AchE, EC 3.1.1.7)和乳酸脱氢酶(lactate dehydrogenase, LDH, EC 1.1.1.28) 的活性变化。结果表明： 处理后1-7 d, 即时浸酸处理的滞育卵, SOD活性由56 517.00 U/g提高到81 986.94 U/g, CAT活性由14.98 U/g提高到106.90 U/g, PK活性由25.19 U/g提高到181.70 U/g, AChE活性由17.88 U/g提高到287.86 U/g, 而LDH活性由169.96 U/g下降到122.82 U/g。 而在非滞育卵中, SOD活性由86 417.99 U/g下降到66 024.19 U/g, LDH活性由169.07 U/g下降到135.02 U/g; CAT活性由1.47 U/g提高到44.37 U/g, PK活性由20.56 U/g提高到92.09 U/g, AChE活性由21.40 U/g提高到99.17 U/g。在滞育卵中, SOD和AChE活性较稳定; CAT活性随发育上升, 而LDH活性随发育而下降; PK活性在胚胎发育的前 4 d呈上升趋势, 随后基本保持稳定。通过了解家蚕滞育卵、 非滞育卵与即时浸酸卵的相关酶活性在胚胎发育过程中存在的变化, 有助于进一步揭示家蚕滞育的机理。 相似文献
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Tao Jiang Jiashuang Li Ping Qian Peng Xue Jin Xu Yanrong Chen Juan Zhu Shunming Tang Qiaoling Zhao Heying Qian Xingjia Shen 《Molecular reproduction and development》2019,86(12):1981-1992
N6‐methyladenosine (m6A) plays a key role in regulating gene expression in myriad organisms. Diapause is an important plastic phenotype that allows insects to survive under specific environmental conditions. However, the diapause molecular mechanism remains unknown. In this study, we analyzed the phylogenetics of genes related to the m6A modification complex in the silkworm (Bombyx mori) based on identified sequences from other organisms. We detected the expression of these genes during different developmental phases from four strains with different voltinism. We also determined total m6A content in cells treated with different diapause hormone concentrations or eggs exposed to hydrochloric acid. Our data revealed that m6A‐modification‐related gene expression and m6A content were greater in diapause‐destinated compared to nondiapause‐destined strains. Our findings suggest that m6A modification may provide significant epigenetic regulation of diapause‐related genes in the silkworm. 相似文献
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Previous study showed that diapause in Bombyx mori eggs can be terminated by dechorionation and that activation in the mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) in dechorionated cultured eggs is involved in diapause termination. In the present study, the possible mechanism underlying activation of ERK upon dechorionation was further investigated. Results showed that mechanical injury of diapause eggs without medium incubation also resulted in rapid increase in the phospho‐ERK levels and that injury increased the phospho‐ERK levels at different stages of both diapause eggs and eggs in which diapause initiation was prevented by HCl. Effects of anaerobiosis on dechorionation‐stimulated phospho‐ERK levels showed that the mechanical injury itself but not the dramatic increase in oxygen uptake upon injury is involved in a rapid activation of ERK. Chemical anaerobiosis on dechorionation‐stimulated phospho‐ERK levels and the in vivo effect of anaerobiosis showed that the supply of oxygen also plays a role in ERK signaling. In addition, injury induced the phosphorylation of c‐jun N‐terminal kinases (JNKs) and p38 kinase, components of two parallel MAPK pathways. A kinase assay showed a dramatic increase in JNK kinase activity in egg lysates upon injury. When newly hatched first instar larvae were injured, an increase in the phospho‐ERK levels similar to that in dechorionated eggs was observed. From the results, we hypothesize that the injury‐induced rapid activation of MAPK signaling, which serves as a natural signal for embryonic development, is related to diapause termination in dechorionated eggs. 相似文献
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For the bivoltine (Dazao) strain of the silkworm Bombyx mori L., diapause expression in progeny is induced by exposure to conditions of 25 °C and continuous illumination (LL) during the maternal generation, whereas an environment of 15 °C and constant darkness (DD) results in nondiapause progeny. Initiation of diapause in progeny can be prevented by treatment of diapause‐programmed eggs with hydrochloric acid (HCl) at approximately 24 h post‐oviposition. To investigate whether glutathione is involved in the regulation of diapause induction and initiation in this species, measurements of total glutathione, reduced glutathione (GSH), oxidised glutathione (GSSG), GSH/GSSG ratio, glutathione S‐transferase (GST) and peroxiredoxins (Prdx) are compared in eggs incubated under LL and DD conditions, and between diapause eggs and those treated with HCl. Compared with DD, eggs incubated under LL have higher total glutathione (GSH + 2GSSG), lower GSH, higher GSSG, a lower GSH/GSSG ratio, lower GST activity and higher Prdx activity at stages 20–25 of maternal embryogenesis. The lower ratio of GSH/GSSG is indicative of pro‐oxidative conditions during diapause induction, which may result from the stronger oxidation of GSH. Compared with HCl‐treated eggs, diapause eggs have lower total glutathione, no difference in GSH, lower GSSG, a higher GSH/GSSG ratio, no difference in GST activity and lower Prdx between 36 and 72 h post‐oviposition. The higher ratio GSH/GSSG is indicative of reducing conditions during diapause initiation, which may a result of the weaker oxidation of GSH. Moreover, variations of Prdx and GST suggest that Prdx rather than GST plays an important role in the oxidation of GSH during the induction and initiation of diapause. 相似文献
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即时浸酸显著提高滞育性家蚕卵辅酶Ⅰ和Ⅱ含量 总被引：1，自引：0，他引：1
即时浸酸在阻止家蚕Bombyx mori卵滞育发动的同时, 提高了其呼吸耗氧量, 抑制了山梨醇积累。本研究利用HPLC法测定了家蚕滞育卵和5 min即时浸酸滞育性卵中辅酶Ⅰ和Ⅱ含量。结果表明： 产下后24-72 h, 家蚕滞育卵中NAD, NADH, NADP和NADPH含量分别下降了30%, 37%, 50%和4%; 而即时浸酸滞育性卵中分别增加了77%, 46%, 142%和241%。不过, 即时浸酸并未显著改变滞育性家蚕卵中NADH/NAD和NADPH/NADP比值。据此推测, 即时浸酸提高滞育性家蚕卵辅酶Ⅰ含量与其呼吸耗氧量增加有关; 即时浸酸显著提高辅酶Ⅱ含量与山梨醇积累抑制无关, 而主要与生物合成加强有关。 相似文献
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Previous study showed that exogenously applied recombinant thymosin from Bombyx mori (BmTHY) reduces B. mori nucleopolyhedrovirus (BmNPV) proliferation in silkworm. Which stands to reason that BmTHY in B. mori is crucial for the defense against BmNPV. However, little is known about the effect of endogenously overexpressed or repressed BmTHY on B. mori resistance to virus infection. To study this issue, we constructed an overexpression and inhibited expression systems of BmTHY in BmN cells. The viral titer and the analysis from the quantitative real‐time polymerase chain reaction (PCR) revealed that overexpression of BmTHY decreased the copies of BmNPV gene gp41, which goes over to inhibit the proliferation of BmNPV in BmN cells, while the inhibited expression of BmTHY significantly enhanced viral proliferation in infected BmN cells. These results indicated that endogenous BmTHY can inhibit BmNPV proliferation and replication in infected BmN cells. Furthermore, Co‐IP showed that BmTHY could bind to actin in BmN cells. Also, the overexpression or inhibited expression of BmTHY shifted the ratio of F/G‐actin in infected BmN cells. Lastly, the BmTHY, an actin‐interacting protein, might be one of the key host factors against BmNPV, which inhibits viral proliferation and replication in BmN cells. 相似文献