Expression Profiles and Response to Exogenous Hormones of β-Fructofuranosidase Gene BmSuc1 in Silkworms(Bombyx mori)

[Objectives]In order to clarify the regulatory effects of insect hormones on the expression of BmSuc1 and provide a reference for further analysis of the function and expression regulation mechanism of BmSuc1,this study explored the expression profiles of BmSuc1 in different tissues and periods of silkworm larvae and the expression changes of BmSuc1 after treatment with exogenous hormones.[Methods]By using the real-time fluorescence quantitative PCR technique,the expression characteristics of BmSuc1 were detected in different periods,different tissues and after treatment with exogenous hormones during the development of silkworm larvae.The expression of BmSuc1 and 20E receptor gene USP was detected after RNA interference with double-stranded RNA(dsRNA)of USP.[Results]The relative expression of BmSuc1 gene in the midgut was the highest,followed by the silk glands,epidermis and hemolymph.However,there was much lower or almost no expression in other tissues.In addition,the BmSuc1 expression profile exhibited a pulse-like pattern in silkworm larvae.The expression level of BmSuc1 was higher at each instar stage before molting,late fifth instar before cocooning and prepupal stage.Silkworm larvae at day 2 of the fifth instar were treated with 20-hydroxyecdysone(20E)and juvenile hormone(JH).It was found that the expression of BmSuc1 was extremely significantly higher at 12 and 18 h after 20E treatment than the control group injected with 0.1%dimethyl sulfoxide(DMSO)(P<0.01,the same below).But there were no significant difference in BmSuc1 expression between the JH treatment and the control group during the measurement time range(P>0.05).The dsRNA of USP was synthesized in vitro and injected into silkworm larvae at day 3 of the fifth instar.It was showed that the USP relative expression was extremely significantly down-regulated at 24 and 36 h after injection,which indicated that dsRNA interference was successful.RNAi of USP would block 20E signal transduction,and the expression of BmSuc1 was inhibited and significantly down-regulated at 24 and 36 h after injection of dsRNA of USP(P<0.05).[Conclusions]The BmSuc1 expression peaks appeared in the molting of silkworm larvae and the metamorphosis of larvae to pupae,which suggests that BmSuc1 may be involved in the metamorphic development process of silkworms.Treatment with exogenous ecdysone 20E can activate the expression of BmSuc1,but blocking the 20E signal transduction pathway may suppress expression of BmSuc1.It indicates that BmSuc1 as a downstream target gene in the 20E signal transduction pathway is directly or indirectly regulated by 20E signals.

Screening and Cloning of RAPD Marker of Fluoride Tolerance Gene in Silkworm,Bombyx mori

In this study,silkworm strain T6,tolerant to fluoride,and silkworm strain 733xin,highly sensitive to fluoride,were used to construct the near-isogenic lines.300 random primers were used in RAPD amplification to DNAs of these lines.A molecular marker named S207 was found linked to the fluoride tolerance gene.Examination to F 2 segregated individuals of the above lines verified that this molecular marker was reliable.Subsequently,the molecular marker was cloned into a T vector (pUCm-T) for sequencing.Comparing with sequences available in the GenBank showed that this molecular marker was novel.We plan to convert it into a SCAR marker to facilitate establishment of a molecular marker assisted breeding system.

Study on Key Technique of Artificial Insemination for Silkworm,Bombyx mori

[Objective] The aim of this study was to investigate the efficient technique of artificial insemination for silkworm. [Method] Sperms were extracted from bursa copulatrix of female moths mated for 30 min through extruding and centrifugal method, and then the semen was injected into other virgin moths with trypsinase. [ Result] A high-effective collection technology of spermatids from silkworm was established successfully, 50 μl semen could be collected by only one person in each hour. The survival rate of spermatids was over 80% in vito after collected from bursa copulatrix, while the obtained semen was quite pure and the average fertilization rate of silkworm was 76,5%. [ Conclusion] The establishment of high-effective semen extraction technique of silkworm provides the technical basis for studies on other related techniques for silkworm sperm.

Molecular Systematic Studies on Chinese Mandarina Silkworm (Bombyx mandarina M.) and Domestic Silkworm (Bombyx mori L.)

Molecular systematic studies on mandarina silkworm (Bombyx mandarina M.) in 11 regions in China and 25 representative strains of domestic silkworm (Bombyx mori L.) were conducted using molecular biology techniques. Results obtained from the analysis of DNA polymorphism and clustering of all the silkworm samples provide new evidence for the view that the domestic silkworm originated from the Chinese mandarina silkworm. On the basis of literature reviewing, a new hypothesis on the origin of the domestic silkworm was put forward. It was thought that the domestic silkworm was most probably domesticated from the Chinese mandarina silkworm of different ecotypes including monovoltinism, bivoltinism and multivoltinism; and that the domestic silkworm had the genetic background of monovoltinism, bivoltinism and multivoltinism at the very beginning of the domestication. The current strains of the domestic silkworm of different voltinism are the evolutionary results of thousands of years of rearing and artificial selections.

Targeting of human aFGF gene into silkworm,Bombyx mori L. through homologous recombination

The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL 1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.

Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bombyx mori) on Day 1 and Day 4 in the 5th Instar Stage

The posterior silk gland （PSG） of silkworm is an important organ where fibroin is synthesized and secreted exclusively. Because fibroin constitutes 75-80% of the silk filament, the mechanism governing fibroin secretion, quality and yield of cocoon can be elucidated by the study on the PSG. Using two-dimensional gel electrophoresis （2-DE） and image analysis system, the changes in the protein composition in the PSG cell were investigated on the day 1 （D1） and day 4 （D4） in the 5th instar stage from five different strains of silkworm （Bombyx mori）. While differences at protein level between days and strains were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse among the different strains. The results suggest that the secretion of fibroin is regulated by multiple proteins. The site of regulation and the proteins responsible for the regulation vary with the strain, which leads to differences between strains in the capacity of fibroin secretion in the PSG cell.

Multiple Interval Mapping for Whole Cocoon Weight and Related Economically Important Traits QTL in Silkworm(Bombyx mori)

A backcrossed population （BC1） derived from a cross between C100 and Dazao was obtained. The quantitative trait loci （QTLs） of the economically important traits for whole cocoon weight, cocoon shell weight, ratio of cocoon shell and weight of pupae, etc., were analyzed for the first time using the multiple interval mapping software WinQTLCart2.0. In total 40 QTLs were detected and contributed to 21 groups based on the constructed linkage map. According to the mapping results, 2, 2, 3, and 2 major QTLs explained over 20% of total phenotypic variations, whereas four QTLs, namely qCW-19, qSW-2, qCSR-4, and qPW-23, explained more than 30% of total phenotypic variations for whole cocoon weight, cocoon shell weight, ratio of cocoon shell and weight of pupae, respectively. Correlated traits QTLs often share the same location. Furthermore, most of the detected QTLs were closed to one-side marker. By using the very closed markers, positive QTLs can be aggregated, which can form a basis for molecular marker-assisted selection and breeding.

Resveratrol elongates the lifespan and improves antioxidant activity in the silkworm Bombyx mori

A number of research has shown that the plant polyphenol resveratrol,one of the most prominent small molecules,has beneficial protective effects in multiple organisms,including worms,flies,and killifish.To understand the effects of resveratrol on lifespan,we evaluated its effects in the silkworm Bombyx mori.In this study,we found that lifespan was significantly prolonged in both female and male silkworms treated with resveratrol.Silkworm larval weight was significantly increased from day 3 of the 5th larval instar(L5D3)to day 7 of the 5th larval instar(L5D7).However,the weight of the pupa,cocoon,and total cocoon was not significantly different in female silkworms with resveratrol treatment than that in controls.Meanwhile,resveratrol significantly improved the thermotolerance of the silkworms,which enhanced their survival rate.Moreover,antioxidant activity was increased by resveratrol in both female and male silkworms.Furthermore,an antioxidant-related signalling pathway,SIRT7-FoxO-GST,was activated in silkworms with resveratrol treatment.Collectively,these results help us to understand the molecular pathways underlying resveratrol induced pro-longevity effects and indicate that silkworm is a promising animal model for evaluating the effects of lifespan-extending drugs.

Cloning and Expression Profile of Deoxyhypusine Snyhtase Gene and Deoxyhypusine Hydroxylase Gene in Silkworm, Bombyx mori

Deoxyhypusine snyhtase （DHS） and deoxyhypusine hydroxylase （DOHH） are the two enzymes that catalyze the synthesis of hypusine within eukaryotic initiation factor 5A （eIF5A）. Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we described the cloning and expression of two full-length cDNAs, encoding respectively DHS-like protein and DOHH-like protein from Bombyx mori by using the methods of bioinformatics, RACE, and RT-PCR technology, named as BmDHS and BmDOHH. Sequencing results indicate that they are 1 311 and 1 874 bp in length including complete open reading frame （ORF） 1 116 and 915 bp, which encode 371 amino acids （molecular weight is about 41.11 kD and isoelectric point is 5.84） and 304 amino acids （molecular weight is about 34.30 kD and isoelectric point is 4.86）, respectively. BmDHS contains only 1 exon, and BmDOHH contains 4 exons and 3 introns. The deduced amino acid sequence of BmDHS contains a deoxyhypusine synthase domain from 47 to 361 amino acid residues, and the deduced amino acid sequence of BmDOHH contains 6 E-Z type HEAT repeat domains （23-52, 54-83, 87-116, 177-206, 208-237, and 241- 270）. Compared to DHS and DOHH amino acid sequences from other species, such as Homo sapiens and Drosophila melanogaster, both silkworm DHS protein and DOHH protein have more than 55% identity. The conservative regions are very similar with each other. The phylogenetic tree analysis indicated that not only DHS but also DOHH from different species has genus-specific features. The expressions of BmDHS and BmDOHH are no tissue and stage specific in our tested samples.

Cloning and characterization of nanos gene in silkworm Bombyx mori

Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5＇-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mor/and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5.38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee （Apis mellifera）. This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.

Evaluation of Impact of Pollen Grains from Bt,Bt/CpTITransgenic Cotton and Bt Corn Plants on the Growth andDevelopment of the Mulberry Silkworm,Bombyx moriLinnaeus (Lepidoptera:Bombycidae)

The S-endotoxin genes of Bacillus thuringiensis (Bt) and proteinase inhibitor (PI) genes are two kinds of genes popularly used for developing transgenic plants resistant to insect pests. To clarify whether there is any risk concerning the effects of pollens from these transgenic crops on non-target insects with economic importance, such as the effects on the growth and development as well as cocoon quality of the silkworm, Bombyx mori Linnaeus, a series of feeding experiments were conducted, using pollens from transgenic cotton or corn containing cry 1Ac, cry1A+CpTI or crylAb genes, compared with pollens from non-transgenic normal cotton and corn as well as the non-pollen treatment. In contrast to the latter ones, pollens from transgenic plants showed no significant adverse effects on larval mortality, cocoon weight, pupa weight, cocoon shell weight, pupation rate, emergence rate and fecundity of the silkworm after neonates were fed with the pollens for 72 h. In addition, no dosage effects of pollens were found. Though the duration of 1st instar larvae was prolonged in the case of feeding with transgenic pollens as compared with those of the non-pollen treatment , but they were not significantly different from those fed with pollens from non-transgenic cotton or corn. Meanwhile, the body weight of the 3rd instar molters fed with transgenic pollens was obviously different from those for non-pollen treatment, and was all significantly heavier than that of the controls. Consequently, it is considered that the adverse effect of pollens from transgenic insect-resistant cotton and corn on the growth and development of the silkworm is negligible.

Expression and subcellular localization of a novel gene <i>Bm-X</i>of the silkworm, <i>Bombyx mori</i>

According to the large-scale sequencing of cDNA library from silkworm pupae, the cDNA of a novel gene with blank research background was identified and temporarily named Bm-X. The length of this cDNA is 778 bp. We obtained its ORF for further study by bioinformatics analysis. It is 444 bp and encodes 147 amino acid residues, with a predicted molecular weight (MW) of 16.4 kD and an isoelectric point (pI) of 3.69. In this study, we successfully constructed a recombinant plasmid pET-28a(+)-Bm-X and expressed it in Escherichia coli. We used the fusion protein rBm-X which purified by Niaffinity chromatography to produce polyclonal antibodies against Bm-X for Western blot analysis. The analysis revealed that Bm-X was expressed in the larval midgut, the epidermis and the silk gland. In addition, the subcellular localization analysis of silkworm ovary epithelial cells (BmN cells) showed that Bm-X protein was located both in cytoplasm and nucleus, and the signal was stronger in cytoplasm than in nucleus. Our findings indicate that Bm-X gene is a novel species-specificity gene and its expression product can be detected in tissues of the fifth silkworm instar larvae and BmN cells.

BmApontic is involved in neurodevelopment in the silkworm Bombyx mori

The nervous system of the silkworm is vital for the development of organisms.It achieves and maintains normal life activities by regulating the function of the organs and all kinds of physiological processes in the silkworm.BmApontic(BmApt),as an imports nt bZIP tran scripti on factor,is required for the formatio n of pigme ntation in the silkworm.However,the fun ction of BmApt in the development of the nervous system of the silkworm remains unclear.Here,we showed that amino acid seque nee of BmApt was evoluti on arily con served in its Myb/SANT motif and basic DNA bindi ng domain.BmApt was expressed in the nervous system at the embry onic stage.Knockdow n of Bmapt by RNA interfere nee resulted in abno rmal development of axons.Moreover,the expression of BmnetrinA,BmnetrinB and Bmfrazzled was decreased in the Bmapt knockdown embryos.These results dem on strate that BmApt controls neurodevelopme nt by activati ng the expressi on of Bmnetrin and Bmfrazzled.

Polymorphism identification of carotenoid binding protein gene transcription in the silkworm, <i>Bombyx mori</i>

Carotenoids play important and diverse roles in insects and their uptake and transport rely on carotenoid binding protein (CBP). The study excavated a cluster of CBP-like transcripts, including full CBP from all of the six yellow cocoon Bombyx strains investigated. Sequencing of 54 cDNA clones revealed 17 different types of transcripts which derived from alternative splicing of CBP gene locus. Five of the novel transcripts were similar with spatial and temporal distribution patterns to CBP, but their expression levels were relatively lower. The author disclosed two more novel alternative spliced transcripts with different transcription start sites from CBP in the 5’ UTRs as well as 11 SNP sites neighboring intron 1 after amplification and sequencing. qRT-PCR analysis gave evidence that relatively more mRNA was transcribed from A-type CBP gene than that from B-type in tissues like silk gland and midgut. Sequences of A- and B-type CBP genes were different in length of domains neighboring the 5’ UTR, thus their mRNA varied both in quantity and transcript types. The SNPs surrounding intron 1 can serve as stable markers to distinguish transcripts from the two isoforms, and they can be used for molecular marker assisted selection.

Critical analysis of correlation and heritability phenomenon in the silkworm, <i>Bombyx mori</i>(Lepidoptera: Bombycidae)

Correlation and heritability studies of quantitative traits are a pre-requisite for judicious selection for genetic improvement of complex characters of economic importance. The success of selection is governed by the degree to which the desired trait is transmitted to the succeeding generation. The nature of selection is to be given due consideration at appropriate developmental stages for pursuing selection in desired direction while improving or evolving high productive breeds or hybrids of the silkworm. Characters showing high heritability as well as high genetic advance respond better to simple phenotypic selection while those having low heritability and low genetic advance may respond better to mass selection. Characters showing high heritability and low genetic advance may yield good response to hybridization and recurrent selection. An attempt has been made in this review article to briefly discuss the magnitude of correlation and heritability in selection strategies for the improvement of quantitative traits in desired direction in the silkworm, Bombyx mori (L.).

Prevalence of Muscardine Disease in Different Silkworm Hybrid, Bombyx Mori L. under Agro-climatic Conditions in Aurangabad （M.S.）, India

A study was carried out in Dr. Babasaheb Ambedkar Marathwada University Campus Aurangabad, Maharashtra State, India for analyzing the prevalence of Muscardine disease during rainy, winter and summer season in bivoltine x bivoltine hybrids viz. CSR2 × CSR4, CSR4 × CSR2 and multi × bivoltine hybrid PM × CSR2. Observation on Muscardine disease was recorded till the onset of spinning. Analysis of results shows that disease prevalence was more in bivoltine x bivoltine hybrids compare to multi × bivoltine hybrids. PM × CSR2 was found to be more resistant towards Muscardine disease compared to other hybrids under agro-climatic conditions of Aurangabad.

Analysis of bulked segregants to identify molecular markers linked with cocoon weight and cocoon shell weight in the silkworm Bombyx mori L

Two silkworm strains viz, B20 A (high cocoon shell ratio) and C.Nichi (low cocoon shell ratio) were sib mated for 10 generations to determine the homozygosis. Both bulked segregant analysis(BSA) and near isogenic lines (NIL) studies were done to identify the RFLP markers closely linked to cocoon shell parameters. Three hundred and fifty two random clones were identified as the low copy number sequence and used for identification of Restriction Fragment Length Polymorphic (RFLP) marker linked to cocoon weight and cocoon shell character. In the bulk segregant analysis, DNA from the parents (B20 A, C.Nichi), F 1 and F 2 progeny of high shell ratio (HSR) and low shell ratio (LSR) were screened for hybridization with the random clones. Polymorphic banding pattern achieved through southern hybridization with different probes indicated the probable correlation of polymorphism with high and low cocoon shell character which are possible landmarks in identifying the putative marker(s) for the cocoon shell character. Out of the 100 probes tried with parents, F 1, F 2 and their bulks, 10 probes were found to be closely linked to cocoon shell characters.

丁烯氟虫腈对家蚕(Bombyx mori)的急性毒性与风险评价

采用食下毒叶法、药膜法和点滴法测定丁烯氟虫腈对家蚕的急性毒性,并进行了急性风险评价。食下毒叶法结果表明丁烯氟虫腈对蚁蚕、2龄、3龄、4龄和5龄起蚕的摄入LC50值(96h,下同)分别为280(227～343)、578(507～652)、3612(3178～4139)、6790(6045～7875)、7151(5932～8705)mg·L-1,均大于200mg·L-1,属于低毒级;桑叶浸药时间为1s、10s、1min、10min和60min时,丁烯氟虫腈对2龄起蚕的LC50值分别为789(690～913)、578(507～652)、443(382～499)、396(349～447)、254(222～285)mg·L-1;家蚕在丁烯氟虫腈药膜上爬行10、30和60min后,对2龄起蚕的接触LC50值分别为101(86.8～131)、66.7(61.7～74.1)、60.9(51.0～83.4)μg·cm-2,对3龄起蚕的点滴接触LD50值为76.2(70.2～84.9)μg·larva-1。急性风险评价结果表明,丁烯氟虫腈对蚁蚕具有中等风险性,对其他龄期家蚕为低风险。因此,丁烯氟虫腈对家蚕的急性毒性和急性风险均较低。

家蚕(Bombyx mori)不同发育阶段饲料适宜pH值的研究

通过改变人工饲料的ｐＨ值，对蚁蚕、稚蚕和壮蚕进行饲育，以了解家蚕幼虫不同发育阶段对人工饲料ｐＨ值的要求，并研究用不同ｐＨ值的人工饲料饲育１～２龄家蚕对３龄改用桑叶育后生长发育的影响．结果表明：蚁蚕摄食和稚蚕饲育的最适人工饲料ｐＨ值分别为４．３和４．６；壮蚕人工饲料的适宜ｐＨ值范围是４．４～４．７；但当３龄采用桑叶育时，１～２龄中所用人工饲料的ｐＨ值，以４．２～４．