家蚕后丝腺亚细胞组分中核酸和蛋白质的分布

丝心蛋白是家蚕后丝腺的分泌产物。在家蚕五龄早期,后丝腺细胞中迅速形成丝心蛋白合成所必需的一些亚细胞结构,在后丝腺细胞质中粗糙内质网迅速增多,平滑内质网迅速形成空泡状或管状。而且生化测定表明,在五岭早期后丝腺中DNA,RNA、蛋白质和脂肪的含量皆迅速增加,这些为在五龄后期丝心院的合成,即为丝心蛋白信息的转录和翻译,提供亚细胞结构和生化代谢的物质基础(Tashiro等1968;赤井弘1973)。因此有必要对家蚕后丝腺亚细胞组分如细胞核、线粒休、微粒体和105,000g上清液中核酸和蛋白质代谢进行研究。本文初步建立一种分离家蚕后丝腺亚细胞组分的差级离心方法,并测定各亚细胞组分中核酸和蛋白质的含量。     

家蚕丝蛋白的合成和保幼激素的调节

本实验查明家委品种的后丝腺细胞数目为1080个,在透射电镜观察下,后丝腺细胞内质网呈片层状,分布着大量的核蛋白体颗粒,细胞核内染色质成块状。用液面铺展技术显示出染色质的特别结构。每个后丝腺细胞能合成DNA0.6—0.8微克,为原来体细胞数的一百万倍,奠定了后丝腺细胞染色体的多线化的物质基础。第7天后丝腺的RNA为5.4毫克,mRNA为总RNA的1%,占细胞核RNA的2.5%,rRNA的碱基比(G+C)为53.1%,热增色效应为22.5%,融点温度?值为43.7℃。染色质丝DNA的融点温度为70.5℃,碱基比(G+C)为40.5%,并分析了染色质的DNA、RNA、组蛋白和非组蛋白的相对比值。每个后丝腺细胞合成240.6微克丝心蛋白,每个细胞每秒钟可合成6×10⁸个丝心蛋白分子。中丝腺储存的丝心蛋白为后丝腺的二十倍。 经保幼激素类似物处理后,蚕的生长期延长,体重和丝腺重量、后丝腺内蛋白质和RNA含量增高,合成丝心蛋白的功能单位——多核蛋白体在量的方面也有所提高。例如在后丝腺RNA方面,雄蚕第7天增加到6.20毫克,每个后丝腺细胞能合成丝心蛋白243.6微克。因此,我们认为,外源保幼激素能促进RNA的合成,维持后丝腺细胞的正常活动,提高多核蛋白体的合成量,从而增加丝心蛋白的合成。     

家蚕五龄幼虫后部丝腺细胞EST的测定和基因表达谱分析

以C108蚕品种五龄第1天和第5天的后部丝腺为材料, 依靠EST分析探讨了有关后部丝腺细胞合成分泌丝素蛋白基因的表达调控问题. 研究结果发现, 测得的9948条EST序列, 经序列拼接分析得到2861个一致性序列, 其中, 重叠的EST群有911个, 单条序列有1950个; 被注释的一致性序列达1335个, 未被注释的一致性序列达1526个; 55.89%, 共5560条与Mita等人发表的家蚕后部丝腺细胞EST没有同源性; 五龄第1天得到的重叠的EST群数和单条序列数都要比五龄第5天的多约1倍; 重叠的EST群组成大小大于50的基因仅占所有一致性序列的0.5%左右, 基因表达的频率开差非常大, 主要表达的基因是与丝素组成和丝素分泌合成有关的基因; 五龄第5天基因表达量与第1天相比, 丝素重链基因高18倍, 丝素轻链基因高9倍, 丝素P25基因高8倍; 经功能注释分析, 被赋予担负细胞组分功能的基因达508个, 担负酶功能的基因达315个. 结果暗示了五龄第1天的基因表达除了作用于丝腺细胞的生长外, 主要是为丝素蛋白合成分泌作准备, 五龄第5天的基因表达主要是合成分泌丝素蛋白; H链、L链和P25蛋白实际可能并没有按6︰6︰1比率构成复合体, 或H链结构特殊, 不易通过EST技术检测到. 在2861个基因中将有相当部分基因参与丝素蛋白的合成与分泌, 说明家蚕后部丝腺细胞合成、分泌丝素蛋白这一生命过程比以前了解的要复杂得多.     

蜕皮激素与丝蛋白合成:植源性蜕皮激素对家蚕后部丝腺核酸代谢的影响

给家蚕五龄幼虫添食植源性蜕皮激素(以下简称MH),对后部丝腺的核酸代谢都有直接影响,但由此引起的效应却是复杂的,与添食的时期和剂量以及蚕品种等因素有关。五龄每天低剂量(夏蚕0.5微克/头,春蚕2微克/头)连续添食和前期一次添食(2—4微克/头)对后丝腺RNA的合成都有明显的促进作用,并且RNA含量增长高峰是在五龄中期以后;每天较高剂量连续添食和中期一次添食,都表现为抑制作用。添食MH对后部丝腺DNA合成的影响与RNA的变化不尽一致;在每天0.5微克/头连续添食区表现出强烈的促进作用,而其余各处理区都出现明显的抑制。由此认为MH能直接作用于遗传物质DNA,通过调节有关基因的复制或者转录速度,从而对丝蛋白的生物合成产生某种影响。     

家蚕后丝腺细胞染色质的一些特性

在家蚕幼虫发育阶段,丝腺细胞的大小和脱氧核糖核酸(DNA)的含量均有巨大增长,而没有细胞分裂发生,这种DNA含量的增加是由于细胞内整个染色体组型多次全部复制的结果,因此称之为多倍化现象。细胞染色体组型的多倍化现象,也在其他生物种类中存在,可是同丝腺细胞比起来可以说是相差很大。后丝腺和中丝腺每个细胞所含DNA最高量大约是体细胞的20—100万倍。这是与它们制造和分泌大量丝蛋白相一致的。后丝腺细胞是合成丝腺的核心——丝心蛋白的主要部位。为了阐明昆虫激素是如何调节和控制丝心蛋白的合成,需要分离出一定纯度的后丝腺细胞染色质来。后丝腺细胞体积大、细胞核呈树枝状使分离工作困难。我们在这方面作了一个尝试,结果报道如下:     

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。     

昆虫激素调节控制的研究——保幼激素类似物对合成甘氨酸和丙氨酸有关酶系的调节控制

五龄后期蚕丝腺后部的细胞几乎只合成蚕丝的丝心蛋白。保幼激素类似物(JHA)能增加蚕丝心蛋白的生物合成。家蚕和蓖麻蚕丝心蛋白中,甘氨酸和丙氨酸的含量约占75%。我们用ZR515和ZR777分别处理五龄蚕后,观察到丝腺后部和脂肪体中合成甘氨酸和丙氨酸有关转氨酶的活力均有明显增加。(1)用ZR515处理五龄家蚕后,使五龄期延长1～2天,茧层量增加约10%。蚕丝腺后部的丙氨酸-乙醛酸转氨酶,丙氨酸-酮丙二酸转氨酶,谷丙转氨酶和谷天转氨酶活力,JHA组均比对照组增高约10～40%,脂肪体中这些酶活力也相应地比对照组增高约30%,其中丙氨酸-乙醛酸转氨酶活力增加到165%。酶活力增加的持续时间约3～5天。(2)用ZR777处理五龄蓖麻蚕后,丝腺后部和脂肪体中的谷天转氨酶、异亮氨酸吨-α-酮戊二酸转氨酶和丙氨酸-乙醛酸转氨酶活力,均比对照组高。本文认为JHA对蚕丝腺后部和脂肪体中形成甘氨酸和丙氨酸的转氨酶有明显的调控作用,从而为丝心蛋白的合成提供更多的甘氨酸和丙氨酸。这也许是JHA增丝机制的一个重要方面。     

昆虫激素调节控制的研究——保幼激素类似物对合成甘氨酸和丙氨酸有关酶系的调节控制

五龄后期蚕丝腺后部的细胞几乎只合成蚕丝的丝心蛋白。保幼激素类似物(JHA)能增加蚕丝心蛋白的生物合成。家蚕和蓖麻蚕丝心蛋白中,甘氨酸和丙氨酸的含量约占75%。我们用ZR515和ZR777分别处理五龄蚕后,观察到丝腺后部和脂肪体中合成甘氨酸和丙氨酸有关转氨酶的活力均有明显增加。(1)用ZR515处理五龄家蚕后,使五龄期延长1～2天,茧层量增加约10%。蚕丝腺后部的丙氨酸-乙醛酸转氨酶,丙氨酸-酮丙二酸转氨酶,谷丙转氨酶和谷天转氨酶活力,JHA 组均比对照组增高约10～40%,脂肪体中这些酶活力也相应地比对照组增高约30%,其中丙氨酸-乙醛酸转氨酶活力增加到165%。酶活力增加的持续时间约3～5天。(2)用ZR777处理五龄蓖麻蚕后,丝腺后部和脂肪体中的谷天转氨酶、异亮氨酸-α-酮戊二酸转氨酶和丙氨酸-乙醛酸转氨酶活力,均比对照组高。本文认为JHA 对蚕丝腺后部和脂肪体中形成甘氨酸和丙氨酸的转氮酶有明显的调控作用,从而为丝心蛋白的合成提供更多的甘氨酸和丙氨酸。这也许是JHA 增丝机制的一个重要方面。     

20-羟蜕皮素对家蚕后部丝腺细胞超微结构的影响

应用超薄切片和电镜技术,详细观察了蜕皮激素(MH)对家蚕Bombyx mori后部丝腺细胞超微结构的影响.电镜观察表明,家蚕后部丝腺细胞对MH处理极为敏感.一经MH处理,其细胞核的形态结构和细胞质中各种细胞器的发生、发展都呈现出明显的变化,并且与MH处理时期及剂量有关:五龄前期适量MH(4μg/头)处理,能促进与丝蛋白合成有关细胞器的形成与发展,加速腺细胞的成熟生长;较高剂量(8—16μg/头)处理,则导致自噬体的过早发生.五龄中、后期MH处理,一方面促进了粗面内质网等细胞器的进一步发达,另一方面也提高了自噬体的发生数量;处理剂量越高,后一种倾向越突出.这些结果证明,后部丝腺细胞超微结构的变化受MH调节.     

蜕皮激素与丝蛋白合成:植源性蜕皮激素对家蚕丝腺蛋白质合成中3H标记甘氨酸参入活性的影响

用³H标记甘氨酸参人法证明:家蚕后部丝腺中丝心蛋白的大量合成, 是在五龄4日以后, 熟蚕时合成活性最高;中部丝腺中丝胶蛋白的大量合成, 是在五龄5日以后, 以熟前一日合成活性最高.五龄每日低剂量连续添食和前期一次添食植源性蜕皮激素(简称MH), 能促进后部丝腺对³H标记甘氨酸的吸收, 显著提高丝心蛋白的合成活性, 而抑制中部丝腺的吸收, 使丝胶蛋白的合成活性明显下降.本试验结果为阐明外源MH对丝蛋白合成的调节机理提供了直接的证据.     

Exogenous prostaglandin F2alpha as a mediator in the regulation of silkworm growth and silk gland genome

Prostaglandins are locally acting hormones that have remarkable variety of physiological functions. They are rapidly synthesized in several types of vertebrate cells as oxygenated metabolites of arachidonic acid in response to various stimuli. In many insect species they are biosynthesized in fat body and hemocytes mainly in response to bacterial infections. In the present study, we administered synthetic analog of prostaglandin F2alpha, the most prominent of the prostaglandins to the 48 h old fifth instar silkworm, Bombyx mori L. at a single dose of 4 microg per larva to study its effects on the larval growth pattern and silk synthesis. The possible role of PGF2alpha at altering the quantum of silk synthesis by controlling the silk gene expression was also studied. The genomic DNA was isolated from the posterior silk gland on Days 5 and 7 of the fifth instar from the prostaglandin treated and the control larvae and were random amplified with arbitrary primers. The result presented notable variation in the amplified product suggesting the participation of PGF2alpha in the silk biosynthesis controlling the silk gene expression. The feeding period of treated larvae was unaffected while the cocoon characters exhibited considerable improvement. The filament traits also were improved notably in the treated larvae. The participation of PGF2alpha analog in the silk biosynthetic process with its physiological and molecular implications are discussed.     

家蚕(Bombyx mori)主要血浆蛋白质及其基因表达的研究

本文用SDS-PAGE法观察不同发育阶段蚕血液中主要血浆蛋白质sp、30KP浓度的变化;从不同发育阶段的蚕脂肪体提取RNA和poly(A)~+-RNA,在兔网织红细胞系作体外翻译并检测翻译产物。结果表明,5龄蚕脂肪体mRNA合成蛋白质的速率为初蛹的2倍;5龄及初蛹脂肪体30KP mRNA活性的发育变化与其相应蛋白质在血液中的浓度变化一致;sp-1在5龄幼虫脂肪体内的表达及卵黄原蛋白(Vg)在蚕蛹脂肪体内的表达具有雌特异性,其表达和性特异性大体是在前翻译水平被调节的。     

Hormonal effects on carotenoid uptake by the silk gland in the silkworm, Bombyx mori

The carotenoid uptake by the silk gland of the silkworm (Bombyx mori), which occurs only during the middle to late period of the last (fifth) instar in the natural condition, was studied in relation to the hormonal controls. During certain stages of the fourth and last instars, the corpus allatum hormone (JH) was found to inhibit the activation of the absorbing function of the silk gland. The absorbing activity was inactivated, if the activated silk gland was implanted into larva at the late stage of the fourth instar in the presence of the moulting hormone (MH). As more ponasterone-A (ecdysone-analogue) was injected into decapitated larvae, the pigmentation of the silk gland was increased; but injection of a high titre inhibited its activity. It seems that, through serial transplantations, the silk gland inactivated experimentally at the late stage of the fourth instar is reactivated in the presence of MH during the middle to late period of the last instar. The results indicate that MH and JH at each stage control the activity of the carotenoid uptake.     

Interrelationship between the silk gland and other tissues in protein metabolism in the latest larval stage of the silkworm, Bombyx mori

Gamma irradiation at scheduled embryonic stages of silkworm eggs caused some disturbances in biological activities, i.e. cell proliferation of the silk gland, growth of larva and larval tissues, and cocoon production. The source of precursors for silk formation at the latest age of the fifth instar was found to be mainly in the ‘integument’ in its disintegrating state. Radioactivity in the tissues of the control and irradiated larvae labelled with ¹⁴C-glycine or ¹⁴C-leucine was used for analysis. Disturbance of synthesis of silk protein and of fat body protein with gamma irradiation decreased the degrading ability of the ‘integument’ and gut proteins.     

家蚕染色体复制区带的初步显示

该文采用家蚕Bomoyx mori活体注射BrdU结合FPG(fluorochrome photolyusis Giem-sa)显带方法,以生殖腺为材料,成功显示出家蚕有丝分裂中期染色体复制带。由于处于S-期的细胞有早有晚,且同一细胞DNA各片段的复制亦有先后,因此BrdU掺入DNA合成的时间也有所不同,从而可产生出早、中、晚复制带型。BrdU掺入时间早,则会在家蚕部分染色体上出现大面积浅染带纹的早复制带。每一染色体皆有其独特的带纹特征,据此可初步将它与其它染色体相互区分;随着BrdU掺入时间的推后,染色体上会出现深浅交替、丰富的带纹,即中复制带型;至S-期DNA合成晚期掺入BrdU,最终染色体出现以深染带纹为主,浅染带纹仅出现于少数染色体的中部、近中部或端部的晚复制带。     

Death commitment in the anterior silk gland of the silkworm, Bombyx mori

The insect steroid hormone, 20-hydroxyecdysone (20E) triggers the programmed cell death (PCD) of the anterior silk glands (ASGs) of the silkworm, Bombyx mori. We tried to determine the time of commitment to die (death commitment) by examining ASG responses to 20E and juvenile hormone analogue (JHA) in vivo as well as in vitro. The ASGs obtained late on day 6 of the fifth instar completed PCD when cultured with 20E, while the ASGs obtained on day 4 and cultured with 20E did not undergo PCD. The ASGs became competent to respond to 20E at mid-day 5. The ASGs with responsiveness to 20E were not sensitive to JHA, indicating that the ASGs were committed to die before becoming capable of responding to 20E. Topical application of JHA on day 4 suppressed 20E-induced PCD, but that on day 5 failed to do so, indicating that the death commitment might occur between day 4 and 5. We also determined the time of death commitment after allatectomy of the fourth instar larvae, a procedure that induced the precocious PCD. Timed application of JHA and culture of ASGs with 20E in the presence of JHA showed that the ASGs had lost their sensitivity to JHA between 72 and 96 h after allatectomy, i.e. 24-48 h before precocious gut purge in the allatectomized larvae. This result is similar to that obtained in the fifth instar. We conclude that the cellular commitment to die takes place one day before the ASGs become competent to respond to 20E.     

Analysis of ESTs and gene expression patterns of the posterior silkgland in the fifth instar larvae of silkworm, Bombyx mori L.

The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without homology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowledge we have.     

Analysis of ESTs and gene expression patterns of the poste-rior silkgland in the fifth instar larvae of silkworm, Bom-byx mori L.

The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without ho-mology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowledge we have.     

The homeodomain protein PBX participates in JH-related suppressive regulation on the expression of major plasma protein genes in the silkworm, Bombyx mori

In the silkworm, Bombyx mori, major plasma proteins referred to as 30K proteins are the most abundant proteins in the hemolymph of final (fifth) instar larvae. Surgical extirpation of corpora allata, the source of a juvenile hormone (JH), causes rapid accumulation of 30K proteins in the hemolymph of fourth instar larvae. The 30K protein 6G1 (30K6G1) gene was repressed in primary cultured fat body cells treated with a JH analog (JHA), methoprene. To identify the JH response element present in the promoter region of the 30K6G1 gene, we performed transfection analyses of the 5'-deletion mutants of the 30K6G1 gene using primary cultured fat body cells, gel retardation assays and in vivo footprinting analysis. The results from those analyses revealed that a JH response element exists in the sequence between positions -147 and -140. When the promoter construct mutated at positions -143, -142, and -141 was transfected to fat body primary cultured cells, the suppression effect on the reporter gene expression caused by JHA was reduced. Gel retardation assay using specific antibody revealed that a PBX protein binds to the JH response element. Northern blot analysis revealed that the gene expression of Bombyx PBX is enhanced in the fat body cells by JHA treatment. These results indicate that PBX proteins are involved in the JH signaling pathway and play an important role in suppressing 30K protein gene expression in the fat body of B. mori.