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.     

丝蛋白合成和分泌期家蚕丝腺中有氧氧化特性分析

【目的】有氧氧化中葡萄糖(Glc)、丙酮酸(PA)、乙酰Co A(AC)、还原型吡啶核苷酸(NADH)和腺苷三磷酸(ATP)摩尔数的理论比值为1∶2∶2∶10∶30~32,而己糖激酶(HK)、磷酸果糖激酶1(PFK1)、丙酮酸激酶(PK)、丙酮酸脱氢酶(PDH)、柠檬酸合酶(CS)、异柠檬酸脱氢酶(ICDHm)、α-酮戊二酸脱氢酶(α-KGDH)、NADH泛醌还原酶(NCR)、琥珀酸泛醌还原酶(SCR)、泛醌细胞色素C还原酶(CCR)、细胞色素C氧化酶(CCO)和ATP合酶(AS)活性的理论比值为1∶1∶2∶2∶2∶2∶2∶10∶2∶12∶12∶26~28。本研究旨在分析丝蛋白合成和分泌期家蚕Bombyx mori丝腺有氧氧化的特性。【方法】利用分光光度法和高效液相色谱法测定了上述生化指标的变化。【结果】丝蛋白合成和分泌期家蚕丝腺中检测不到Glc,产物含量以1/30 ATP,1/10 NADH,1/2 AC和1/2 PA的顺序递增;糖酵解途径相关酶活性,以PFK1活性最低;三羧酸循环相关酶活性以1/2 ICDHm,1/2α-KGDH和1/2 CS的顺序递增;氧化磷酸化相关酶包括1/26 AS,1/10 NCR,1/2 SCR,1/12 CCR和1/12 CCO的活性以1/26 AS活性最低;1/26 AS,1/2 ICDHm,1/2 PDH和PFK1的活性依次递增。NADH含量、ATP含量、PFK1活性、PDH活性和NCR活性在丝蛋白合成期升高,但在丝蛋白分泌期下降。【结论】据此推测,家蚕丝腺中PFK1,ICDHm和AS分别是糖酵解途径、三羧酸循环和氧化磷酸化的限速酶;糖酵解途径、丙酮酸脱氢、三羧酸循环和氧化磷酸化速率依次递减;有氧氧化速率在丝蛋白合成期升高,相反在丝蛋白分泌期降低。     

DNA synthesis and endomitoses in the giant nuclei of the silkgland of Bombyx mori.

At the first symptoms of organisation of the silkgland in the embryo, mitoses stop and nuclei start to grow. Through autoradiographic studies, performed after sequenced labeling with [3H] and [14C] thymidine, the durations of the different phases of DNA synthesis cycles (T = 42 to 48 h, S = 22 to 25 h, G = 20 to 23 h) are established. These durations are in fact identical during the second and the third instar, and the same, whatever region is concerned : posterior, middle or anterior parts. A model has been established to account for the distribution of the S phases during the second and third instars. The DNA synthesis in all nuclei of a given region has been followed during the first four instars by labelling with [3H]thymidine. The activity goes through maxima and minima, depending on the percent of nuclei synthesizing DNA and their synchronism, both being characteristic of each region; long resting periods are observed during the molting stages of the first three instars in the middle and the anterior parts. The coincidence is obvious between the maxima and minima and respectively the S and G phases of the model. DNA assays agree with the distribution of cycles established by autoradiography if one admits that each cycle does lead to a doubling of the amount of DNA. The overall DNA synthesis from the diploid value is estimated to correspond to 18-19 endomitoic cycles in the nucleic of the posterior part, 19-20 cycles of those of the middle part and 13 in those of the anterior part. The analysis of chromosome structures, by squashing the nuclear content, shows that existence of a complete endomitotic cycle: the doubling of chromosomes is associated with condensed structures, alternating with a decondensed state of chromatin, responsible for the DNA synthesis. The female heterochromatin undergoes a restricted morphological cycle delayed with respect to bulk chromatin. It is characterized by a late DNA duplication and by non dispersed daughter chromosomes. Some of these aspects are, to a lesser extent, reproduced in groups of autosomic chromosomes.     

Structural analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori

To study the minimal length required for the secretion of recombinant proteins and silkproteins in posterior silk gland,the signal peptide(SP)of the fibroin heavy chain(FibH)of silkwormBombyx mori was systematically shortened from the C-terminal.Its effect on the secretion of protein wasobserved using enhanced green fluorescent protein(EGFP)as a reporter.Secretion of EGFP fusion proteinswas examined under fluorescence microscope.FibH SPs with lengths of 20,18,16 and 12 a.a.can directthe secretion of the reporter,yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP wasshortened to 12a.a., the secretion efficiency was decreased slightly and cleavage occurred within EGFP.When 16a.a.of the FibH SP were used,the secretion of fusion protein was normal and the cleavage sitewas between the Gly-Ser linker and Met,the starting amino acid of EGFP. These findings are applicable forthe expression of foreign proteins in silkworm silk gland.The cleavage site of the SP is discussed andcompared with the predictive results of the SignalP 3.0 online prediction program.     

Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori‐type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non‐transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.     

家蚕丝素重链启动子驱动DsRed的瞬时分泌表达

根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,克隆了家蚕丝素重链基因(Fib-H)启动子及其下游的信号肽序列(FibHS),将DsRed基因与信号肽序列融合构建了分泌型瞬时表达载体;转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed;家蚕注射载体后,可在丝腺腔中检测到红色荧光,表明瞬时表达的DsRed分泌到丝腺腔,推测所克隆的序列具有信号肽的功能。此外,本研究为家蚕丝腺生物反应器分泌表达外源基因的研究奠定了基础。     

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 crystal structure of Bombyx mori silk fibroin at the air–water interface

A new crystalline polymorph of Bombyx mori silk, which forms at the air–water interface, has been characterized. A previous study found this structure to be trigonal, and to be distinctly different than the two previously observed silk crystal structures, silk I and silk II. This new structure was named silk III. Identification of this new silk polymorph was based on evidence from transmission electron microscopy and electron diffraction, coupled with molecular modeling. In the current paper, additional data enables us to refine our model of the silk III structure. Some single crystal electron diffraction patterns indicate a deviation in symmetry away from a perfect trigonal unit cell to monoclinic unit cell. The detailed shape of the powder diffraction peaks also supports a monoclinic cell. The monoclinic crystal structure has an nonprimitive unit cell incorporating a slightly distorted hexagonal packing of silk molecular helices. The chains each assume a threefold helical conformation, resulting in a crystal structure similar to that observed for polyglycine II, but with some additional sheet-like packing features common to the threefold helical crystalline forms of many glycine-rich polypeptides. © 1997 John Wiley & Sons, Inc. Biopoly 42: 705–717, 1997     

Study on fibroin heavy chain of the silkworm Bombyx mori by fluorescence in situ hybridization (FISH)

The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized the Fib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified that Fib-H has only one locus, thus providing a temporary solution to the problem about its localization.     

家蚕丝素P25蛋白基因启动子顺式作用元件的功能分析

为了阐明蚕丝蛋白基因表达调控的分子机制, 利用Bac-to-Bac杆状病毒表达系统及实时荧光定量PCR等技术, 对家蚕Bombyx mori丝素P25基因启动子上游1 233 bp的活性及其调控元件的功能进行了分析。结果表明： 在P25启动子上游-423~-1 233区和-127~-238区存在正调控元件, 在-238~-423区段存在负调控元件; PSGF和BMFA两结合元件在P25基因表达中起负调控作用。PSGF结合元件对A3启动子在后部丝腺的活性具有一定的增强作用, 进一步验证了PSGF调控元件的功能。通过对P25基因启动子的活性分析, 尤其是对PSGF和BMFA调控元件的功能分析, 有利于进一步了解P25基因表达调控的精细机制。     

The gene expression profile of Bombyx mori silkgland

During prepupal stage, the genes expression in silkgland is considered as a model for gene expression and regulation of eukaryotes. Aiming to comprehensively interpret gene expression profile in the silkgland, we collected all currently available EST, complete cDNA and protein expression information and other gene expression testing data published before, and explored their roles in their function pathways level. With the analysis of interaction between the known proteins and putative bio-macromolecules partners in silico, we list our prediction results in the form of pathway classification and test some of their expressions by experiments.     

带有丝素重链信号肽序列的家蚕丝胶蛋白启动子驱动DsRed的瞬时分泌表达

为了探讨家蚕Bombyx mori丝素蛋白重链信号肽序列在中部丝腺组织中是否具有功能活性,根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,构建了带有丝素重链基因fib-H信号肽的家蚕丝胶-1(ser-1)启动子(ser-HS),用ser-HS驱动DsRed基因构建了分泌型瞬时表达载体pSK-SerHS-DsRed-polyA。转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed。家蚕注射载体后,可在中部丝腺腔中检测到红色荧光,表明瞬时表达的DsRed已分泌到丝腺腔内。据此提出克隆的fib-H信号肽序列在家蚕中部丝腺组织中具有信号肽的功能。     

Structural characterization and artificial fiber formation of Bombyx mori silk fibroin in hexafluoro-iso-propanol solvent system

High-resolution solution (13)C-NMR and CD studies of Bombyx mori silk fibroin revealed the presence of an ordered secondary structure 3(10)-helix, in hexafluoro-iso-propanol (HFIP). The solid-state structure of the silk fibroin film prepared by drying it gently from the HFIP solution still keep the structure, 3(10)-helix, which was studied with high-resolution solid state (13)C-NMR. The structural transition from the 3(10)-helix to silk II structure, heterogeneous structure including antiparallel beta-sheet, occurred during the artificial spinning from the HFIP solution. The wide-angle x-ray diffraction and differential scanning calorimetry thermograms of the artificial spinning fiber after postspinning treatments were observed together with the stress-strain curves. The results emphasize that the molecular structures, controlled morphology, and mechanical properties of the protein-based synthetic polymers can be modulated for enhancing biocompatibility.