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.

Screening of Deltamethrin Resistant Strain of Bombyx Mandarina

[ Objective ] The aim of the study was to explore the resistance development of deltamethrin through selection of dettamethrin resistant strain of wild silkworm （ Bombyx Mandarina）, thus providing the basis for scientific pesticide application and resistance management. [ Method] The wild silkworms collected from three different regions were reared indoors, and the sensitivity of their parents to deltamethrin was detected by topical application. The larvae in each generation were treated with deltamethdn in median lethal dose or so by.topical application. The mortality of larva was analyzed for the establishment of toxicity regression equations and the calculation of the multiple or increased multiple of deltamethdn resistance.[Result] After the Qidong Bornbyx Mandarina （YQD） fed with mulberry leaves were selected for three generations indoors, the multiple of deltamethdn resistance of F, was 14.26, 1.2 times as great as that of Fo ; after the Bombyx Mandarina from mulberry garden of Soochow University （YSD） fed with mulberry leaves were selected for three generations indoors, the multiple of deltamethrin resistance of F4 was 16.48, 1.9 times as great as that of Fo ; after the Wujiang Bombyx Mandarina （YWJ） fed with artificial diets were selected for three generations indoors during six generations, the multiple of deltamethrin resistance of Fe was 18.67, 1.2 times as great as that of Fo. [ Conclusioa] With the selection in same dose, the resistance multiple of YSD increases more rapidly than that of YQD; under double selection of artificial diets and insecticide, the resistance multiple of YWJ increases more slowly than that of YQD.

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.

Expression of Recombinant Gene P1-2A3C of FMDV Asia I in Different Bombyx mori Varieties

[Objective] The aim was to investigate the expression level of recombinant gene P1-2A3C of FMDV Asia I in different Bombyx mori varieties,so as to screen out the B.mori varieties suitable for the foreign gene expression.[Method] The recombinant B.mori baculovirus rBmNPV（P1-2A3C） are injected into pupae of original species and hybrids of B.mori respectively.Then,the expression of antigen is detected by ELISA method and compared.[Result] The expression level of P1-2A3C significantly differed in different B.mori varieties,in which,the hybrids of Qiufeng×TQ78 and Qiufeng×Sijiaojian could be considered as the effective bioreactors for high-level expression of foreign genes.[Conclusion] This study provided the basis for breeding special B.mori varieties which can highly express target protein of Asia I FMDV.

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.

Construction of Midgut Tissue-Specific cDNA Library of Bombyx mandarina M. and Isolation and Sequence Analysis of Serine Protease Gene Fragment

[Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion.

Development and Utilization of Naturally Colored Cocoon Resources of Bombyx mori

This paper summarized the diversity and prominent characteristics of naturally colored cocoon resources of Bombyx mori, and introduced the production methods of colored cocoons, the development and utilization of naturally colored cocoon resources at home and abroad as well as the variety breeding instances, based on which suggestions were proposed for the development of colored cocoon industry.

Breeding of a Natural Colored-Cocoon Bombyx mori Variety Shuhuang No.1

[Objective] This study aimed to develop a new Bombyx mori variety Shuhuang No.1 and investigate its characteristics.[Method] The new variety was developed through cross breeding and pedigree separation.Its characteristics were identified by laboratory tests and rural promotion practices in Sichuan Province.[Result]The cocoon produced by the new variety Shuhuang No.1 was golden-yellow and radiantly beautiful.Shuhuang No.1 was strong and easy to rear,uniform in development.The male and female ones could be distinguished by their larval color and markings.The cocoon yield per 10 000 the 4th instar larvae reached 20.30 kg; the length of a cocoon filament was about 1 115.0 m,and a non-broken filament was 882.95 mm long,accounting for 79.15％ of the total length.The raw silk ratio of fresh cocoon was 18.39％,and the neatness was 91.25 point.The main economic features of Shuhuang No.1 were better than those of the control.[Conclusion] The new variety had been approved by Sichuan Silkworm Evaluation Commission.It can be promoted in parent silkworm rearing areas Sichuan,Chongqing,Yangtze River basin.

Molecular Detection of Healthiness of Bombyx mori

[Objective] The paper was to explore the regularity between heat shock protein expression and the healthiness changes of Bombyx moil materials. [Method] The representative heat shock protein gene Bmhsp24.3 was screened by bioinfor- matic analysis method, and carried out real-time PCR expression analysis. [Result] The target gene Bmhsp24.3 expressed in different B. mori materials, but the expres- sion level in different materials significantly varied. The relative expression level of the gene had different degrees of changes under different rearing conditions. With the increase of rearing temperature, the gene expression was upregulated. The ma- terials with better healthiness had remarkable increase in expression of target gene, while the materials with poorer healthiness had less increase in expression of target gene. The expression difference of target gene Bmhsp24.3 was exactly consistent with the healthiness of breeds. [Conclusion] The healthiness of materials had rela- tionship with expression of target gene Bmhsp24.3. the higher the expression of tar- get gene Bmhsp24.3 was, the better the healthiness of materials was; conversely, the lower the expression of target gene Bmhsp24.3 was, the poorer the healthiness of materials was.

Relationship between Biological Characteristics of Beauveria bassiana (Bals.) Vuill and Pathogenicity to Bombyx mori L.

[Objective] This study was to investigate the relationship between biological characteristics of Beauveria bassiana （Bals.） Vuill and pathogenicity to Bombyx rnori L, with the aim to provide scientific basis for the control of white muscardine in Bombyx mori L. [Method] The strains isolated and purified from the 6 Beauveria bassiana biocontrol agents from all over the country and the 3 white muscardine silkworms collected from Guangxi provincial silkworm rearing areas were identified by the morphological observation and molecular biology technology. The pathogenicity of B. bassaina to silkworms was determined, and the biological characteristics such as growth diameter, sporulation and the extracellular protease activity of the different B. bassiana strains were compared. [Result] The isolated 9 strains were all B. bassaina （Bals.） Vuillemin, and all strains had high pathogenicity to silkworm, but with different pathogenicities. The growth diameter, sporulation and extracellular protease activity of different B. bassiana strains were also different, and showed correlation with the patheogenicity to silkworms. [Conclusion] B. bassiana spores production amount and exocellular protease activity had significant positive correlation with their pathogenicity to silkworm.

Bombyx mori Pyridoxal Kinase cDNA Cloning and Enzymatic Characterization

Pyridoxal kinase （PLK） （EC 2.7.1.35） catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5＇-phosphate （PLP）, an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method （GenBank accession number： DQ452397）. The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 k.Da. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.

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．

壬基酚对家蚕(Bombyx mori)生长发育的影响

采用添加环境激素壬基酚(Nonylphenol,NP)人工饲料饲养家蚕(Bombyx mori)的试验方法,观察了家蚕的生长速度、生长量、变态、生命力情况。结果表明,连续取食0.500mmol·L-1以上浓度壬基酚的饲料,家蚕幼虫和蛹的生长速度、生长量、发育整齐度、生命力等都明显下降,在发育后期和蜕皮、化蛹、羽化等变态时期更加明显。同时,NP在蚕体内可能有累积作用。2.000mmol·L-1浓度的NP能使家蚕幼虫很快死亡。NP对家蚕生长发育的影响有明显的性别差异,导致家蚕的雌雄大小开差缩小,并且具有显著的浓度效应和时间效应,是环境激素效应的典型表现。

家蚕Bombyx mori L.染色体的G带组型研究

以今井的涂片法制备染色体标本,采用GUG法进行G-带分染,研究了家蚕早双线期染色体的组型。依照染色体相对长度及带型鉴别了家蚕28对染色体,并绘制了染色体组型模式图。利用限性普斑系统W 染色体上的易位片断作标识,观察到不对称配对二价体,推论此异形二价体即性染色体。性染色体在14—16染色体范围内。

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.

家蚕(Bombyx mori)血细胞研究进展

家蚕是具有重要经济价值的驯化昆虫,也是完成全基因组测序的具有重要研究价值的鳞翅目模式昆虫。家蚕血液在蚕体的生长发育过程中担负着储存能量、伤口愈合、传递压力、免疫防御等重要生理功能。基于传统的细胞形态学观察方法已经不能对家蚕血细胞的生物学功能进行深入研究。近几年,本实验室通过5-溴脱氧尿嘧啶核苷(Brd U)标记法和细胞特异性的分子标记等方法,对家蚕血细胞的种类、血细胞的增殖发育机制和血细胞的免疫防御功能等进行了较为系统的研究,本文概述在上述几个研究方向中取得的最新进展,为该领域后续深入研究提供理论依据和技术参考。

Studies on the Mutant Systems of the Bombyx mori Gene Bank

Through over ten years of study, more than 1 000 genetic materials including mutant genes, chromosomal variation strains and special genetic materials of Bombyx mori, Linnaeus, collected, introduced or created since 1940s especially late 1980s, have been sorted out and put in order. After identifications and genetic analyses of their morphological, physiological and biochemical characters, the silkworm gene bank was constructed and the preservation system was perfected, and more than 600 silkworm strains were kept in this gene bank. The preserved silkworm mutant genes have covered more than 90% of existent ones across the world, in which, more than 100 are rare and precious mutant genes, and over 60 mutant genes were found and studied for the first time. Through hybrid analyses, linkage tests and three-point gene location tests, a perfect linkage retrieval labeling system of silkworm was established, which included 230 marker genes covering all the 28 linkage groups of Bombyx mori. The gene location system (composite system of recessive genes) of different linkage groups was set up. The intergenic complementation of mutant egg color and third type of maternal heredity egg color have been found, and indicated that the epistatic effect of mutant gene of white egg is universal. Twenty eight independent near isogenic lines marked with morphological mutation gene have been created and a series of novel breeding materials possessing great potential for application such as high feeding efficiency, special sex markers, natural colored silk, resistance to disease, wider feeding range and adjustable parthenogenesis, etc. , have been developed. The sustainable maintenance and management technique system of silkworm gene resources were well established.