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.

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.

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.

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.

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.

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。

家蚕30K蛋白BmLP1的表达模式及其在胚胎中的作用

家蚕30K蛋白是家蚕血液和卵中的高丰度蛋白,参与营养、免疫、抑制细胞凋亡等功能.探究30K蛋白在家蚕体内的表达模式有助于更清楚地了解30K蛋白的功能.利用生物信息学方法发现4种30K蛋白基因Bmlp1,Bmlp2,Bmlp3和Bmlp7都含有信号肽.其中,Bmlp1与其他30K蛋白的序列相似性小于50%.半定量RT-PCR结果表明,Bmlp1从5龄第3 d到化蛹第1 d在脂肪体中表达量都比较高,之后表达量降低.Western blotting结果表明,BmLP1从5龄第5 d到化蛹第4 d在血淋巴中的含量都比较高,之后含量降低.与血淋巴中的变化趋势不同,卵巢中的BmLP1在上蔟第2 d被检测到,之后含量一直增加,化蛾时含量达到最高.这是由于上蔟第2 d卵巢管迅速长大,血液中的BmLP1等营养蛋白逐渐转运至卵巢管的未受精卵并开始积累.免疫组织化学定位结果表明,在胚胎发育前期,BmLP1分布于蚕卵的卵黄颗粒中,在孵化前期,BmLP1被吞入胚胎的肠道内.体外孵育结果表明,蚁蚕粗提物可以降解BmLP1,暗示蚁蚕肠道内可能存在某种蛋白酶可以将大部分BmLP1水解,最终为蚁蚕的孵化提供能量或者氨基酸.系统分析了BmLP1在不同发育时期脂肪体、血液、卵巢和胚胎中的表达特征,揭示了其在胚胎发育过程中的重要作用.

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.

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.

桑蚕(Bombyx mori L.)1～2龄人工饲料1回育3～5龄桑叶育及其成本分析

通过春、夏２期桑蚕１～２龄人工饲料１回育３～５龄桑叶育的农村试养，结果表明，试验区的茧丝质成绩春季基本接近全龄桑叶育，夏季优于全龄桑叶育；通过小蚕人工饲料育的成本分析，采用１～２龄人工饲料１回育每盒蚕种可节省７．５３元。

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.

辛基酚对家蚕(Bombyx mori L.)生长发育的影响

为探讨辛基酚对鳞翅目模式昆虫家蚕生长发育的影响,使用添加0.01~0.08 g·kg-1辛基酚(4-tert-octylphenol,4-t-OP)的人工饲料饲养家蚕(Bombyx mori L.),研究了4-t-OP对家蚕幼虫和蛹的生长发育速度、生长量、发育整齐度和生命力等影响。结果表明:连续取食0.01 g·kg-1以上浓度4-t-OP的饲料,家蚕幼虫和蛹的生长发育速度(幼虫期和蛹期发育经过时间)、生长量(幼虫期体质量和蛹期茧层量)、生命力(3龄起蚕绝食生命时数、4龄起蚕结茧率、蚕茧的死笼率和羽化率)等都明显下降,抑制眠蚕体质量。4-t-OP对家蚕幼虫生长发育后期、化蛹及羽化等发育变态期的影响更加明显,其在家蚕体内可能有累积效应。添食0.08 g·kg-14-t-OP的家蚕不能完成生活史。研究表明,4-t-OP对家蚕生长发育毒性影响有显著的浓度效应和时间效应。

Effect of vertebrate hormones and prostaglandins on growth, silk quality and metabolic activities of Bombyx mori L

The economic importance of silkworm has moved biologists to explore various intricate mechanisms of the action of vertebrate hormones. The dietary administration of several vertebrate hormones and prostaglandins enhanced both developmental and metabolic processes of silkworm, Bombyx mori L. The main objective of sericulture research is to apply the results to achieve superior quality silk and greater output, to apply lab findings to achieve desirable ecenomic results.

Detection of Contamination and Analysis of Vertical Transmission of BmNPV in Eggs and Moths of Bombyx mori

This study reports the molecular detection of Bombyx mori nucleopolyhedrovirus (BmNPV) in silkworm strains of the Universidade Estadual de Maringá Brazilian Germplasm Bank (UBGB). DNA extraction was carried out by using six Bombyx mori female moths of each strain, followed by PCR amplification. A pair of primers was designed based on a specific sequence of the baculovirus genome related to the BmNPV ORF 14. Another pair of primers was used to amplify the silkworm Actin A3 gene segment, which was used as positive control. Twenty gene pools were analyzed, and fifteen revealed a fragment of 443 base pairs (bp), which indicated the presence of the BmNPV. The frequency of contaminated moths was as following: 100% for silkworm strains M18-2, M12-2 and J1;83% for C25, C75 and C24 strains;66% for KR01;50% for M11-A;33% for AS3, B106, M8 and M11 and 16% for C211, E8 and Hindu strains. These are promising results for the identification of contaminated B. mori moths by BmNPV, which may prevent virus proliferation in subsequent generations. We also analyzed DNA samples extracted from B. mori eggs, but the results were not conclusive regarding the detection of the fragments of the expected size (443 bp). The difficulty in detecting BmNPV contamination in B. mori eggs may be due to the low concentration of virus in samples.

1-Deoxynojirimycin Content and Alfa-Glucosidase Inhibitory Activity and Heat Stability of 1-Deoxynojirimycin in Silkworm Powder

Silkworm powder containing 1-deoxynojirimycin (DNJ) has α-glucosidase inhibitory activity and is promising as a complementary and alternative medicine (CAM) agent in Japan. Silkworm powder produced in Korea was extracted with 75% ethanol. The extract was derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl), and DNJ-FMOC content was measured by HPLC. Then, alfa-glucosidase inhibition by silkworm and mulberry powder in pig liver crude enzyme was assayed using 4-nitrophenyl-alfa-D-glucopyranoside as a substrate. Silkworm powder DNJ content (0.39 to 0.58%) was higher than that in mulberry powder (0.08 to 0.12%). The alfa-glucosidase inhibitory activity of silkworm powder was more potent than that of mulberry leaves and green tea. Silkworm powder DNJ was stable upon heating to 121?C for up to 15 min.

Analysis of SSR Fingerprints in Introduced Silkworm Germplasm Resources

Thirty-five SSR markers were used to construct 96 silkworm races fingerprint. All the SSR markers were polymorphic and unambiguously separated silkworm strains from each other. A total of 467 alleles were detected with a mean value of 13.34 alleles/locus （range 3-28）. The mean polymorphism index content （PIC） was 0.71 （range 0.299-0.919）. UPGMA cluster analysis of Nei＇s genetic distance grouped silkworm strains on the basis of their origin. The results indicated that SSR markers are efficient tools for fingerprinting cultivars and conducting genetic diversity studies in the silkworm.

The Mulberry Silkworm--A New Source of Bioactive Proteins

Beyond the commonly known proteins built silk fibres--fibroin and sericin, there are almost 300 bioactive proteins in the silkworm haemolymph. The aim of this work was to present bioactive compounds obtained from the silk fibre and isolated from the body of this insect, which may be used in medical and pharmacological applications. The most important are bioactive proteins. However, the juvenile stages of mulberry silkworm possess other very valuable active substances.

家蚕与桑树相互作用的研究现状及进展

栽桑养蚕是世界农耕文明的一大壮举。专食桑叶的家蚕由其祖先野桑蚕经人工驯化而来,是第一个完成全基因组测序的鳞翅目昆虫。本文综述家蚕与桑树协同进化方面的研究证据,包括家蚕蛋白酶与桑树蛋白酶抑制子的互作、家蚕对桑树次生代谢物质的适应以及桑树尿素酶对家蚕氮代谢的意义等,为今后深入解析家蚕与桑树的互作提供新见解,并丰富植食性昆虫与宿主植物互作的研究信息。

家蚕体内细菌感染模型建立及不同菌株的协同致病特征分析

【目的】建立家蚕体内细菌感染模型,揭示不同细菌协同感染家蚕的致病力,明确云南蚕区家蚕细菌病的发病原因及其规律,为指导家蚕生产过程中细菌病的防治提供科学依据。【方法】从家蚕龄期、感染浓度和感染方法3个因素出发构建家蚕体内细菌感染模型,然后选用从云南地区自然发病蚕体中分离获得的5株肠道致病菌[Mammaliicoccus sciuri(葡萄球菌属)、粘质沙雷氏菌(Serratia marcescens)、弗氏柠檬酸杆菌(Citrobacter freundii)、Klebsiella variicola(克雷伯菌属)和Pseudomonas soli(假单胞菌属)],开展5种单菌感染及17种组合协同感染试验。【结果】通过建立家蚕体内细菌感染模型发现,家蚕群体添食细菌比单头添食的存活率低;细菌感染浓度选择时建议二龄期家蚕感染选用5.0 OD/mL、四龄期和五龄期家蚕感染选用10.0 OD/mL。选取的5株细菌对家蚕均具有致病力,整体上表现为随着细菌感染时间的延长,家蚕存活率逐渐降低;小蚕期感染细菌对家蚕的影响更明显,至五龄期有大量家蚕发病死亡,与养蚕过程中的细菌病发生规律相同。无论是小龄期感染还是大龄期感染,细菌协同感染的家蚕存活率总体上低于单菌感染,尤其是粘质沙雷氏菌参与的协同感染,至上蔟期的家蚕存活率均较低。此外,无论是二龄期感染还是四龄期感染,夏季家蚕的存活率总体上均高于春季家蚕,说明云南蚕区夏季家蚕的抗病力优于春季家蚕。【结论】5株分离自云南地区自然发病蚕体的病原菌对家蚕产生明显致病性,其致病性与感染龄期、感染浓度、不同细菌组合及感染季节等均有关,总体上具体表现为小蚕期感染较大蚕期感染对家蚕的影响更明显,细菌协同感染比单菌感染的危害更严重,且夏季家蚕感染后的存活率高于春季家蚕。