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.

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.

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.

关于桑蚕(Bombyx mori L.)对人工饮料摄食性的研究──Ⅰ.不同收蚁时间和饲料含水率对小蚕发育的影响

为解决发育不齐的问题，就家蚕不同收蚁时间、不同饲料含水率对家蚕幼虫发育的影响进行了研究。结果发现：①发育整齐度，孵化当天收蚁＞二夜包＞三夜包＞四夜包；②饲料含水率以73％左右为适当．过高或过低都会延缓蚕的发育，影响发育整齐度。

Proboscipedia and Sex combs reduced are essential for embryonic labial palpus specification in Bombyx mori

Hox genes of proboscipedia(pb)and Sex combs reduced(Scr)and their cofactors are required for determi nation of in sect mouthpart identity and play important roles in in sect orga n morphoge nesis.However,i n the lepidoptera n in sects,in cludi ng many agricultural and forest pests,the roles of these genes in determi nation of mouthpart morphogenesis are un clear.Here we report that both BmPB(Gene ID:101740380)and BmSCR(Gene ID:692761)are essential for labial palpus specification in a lepidoptera n model in sect,the silkworm,Bombyx mori.During embry onic morphoge nesis,CRISPR/Cas9-mediated mutagenesis of BmPB induced transformation of labial palpus into thoracic leg and BmSCR mutation transformed labial palpus into maxilla.Mutagenesis of Hox-related cofactor extradenticle(BmEXD;Gene ID:692478)and its nuclear localization factor homothorax(BmHTH;Gene ID:576938425)also induced distortion of embryonic labial palpus.Furthermore,mutagenesis of each of the four genes induced severe degeneration of spigot morphogenesis,an important part of spinneret structure.Quantitative real-time PCR analysis further revealed that the transcriptional interaction among these genes.Our data thus provide no vel evidence for Hox gene regulati on of in sect embry onic patterning.

A Study on the Activity of Carboxylesterase and the Differential Expression of Its Gene in the Midguts of Bombyx mori Resistant to BmDNV-Z

This study was to discuss the relationship among the change in the activity of Bombyx mori carboxylesterase （BmCarE） in the midguts, the differential expression of BmCarE gene （bmcare） in the midguts, and the ability of Bombyx mori resistant to densonucleosis virus （BmDNV）, and to elucidate the molecular mechanism of resistance to BmDNV-Z. With two silkworm strains, HUABA, which is susceptible to BmDNV-Z, and BC8 （a near isogenic line of HUABA）, which is completely resistant to the same virus, as materials, the activity of BmCarE in the midgut was determined by Bio-Tek Synergy, and the differential expression of bmcare between the two strains was investigated by real-time fluorescence quantitative PCR, both at 12, 36, and 72 h post oral inoculation of the two strains with virus （hereafter referred as inoculation）. While the activity of BmCarE in the midguts of BC8 inoculation group at 12 h post inoculation was higher than that in the BC8 control group, the HUABA inoculated group, and the HUABA control group by 3.28, 2.26, and 3.02 times, respectively, with the difference being highly significant （P 〈 0.01）, there was no statistical difference among the other groups. The relative expression level of bmcare in the midguts of BC8 inoculation group at 12 h post inoculation was higher than that in the BC8 control group, the HUABA inoculation group, and the HUABA control group by 17.714, 21.76, and 15.09 times, respectively, with the difference being highly significant （P 〈 0.01）, and there was no statistical difference among other groups. The elevation of BmCarE activity and expression level of bmcare in the resistant strain at 12 h post inoculation may relate to the resistant gene （nsd/nsd） and the stimulation of BmDNV-Z. The molecular basis for the elevation of BmCarE activity in the resistant strain BC8 may be the change in the expression level of bmcare.

关于桑蚕(Bombyx moriL.)对人工饲料摄食性的研究 Ⅱ.饲料的简易化制作和同一蚕品种对人工饲料摄食性的个体差异

为解决人工饲料育蚕发育不齐问题 ,就饲料的不同调制方法对桑蚕幼虫发育的影响以及同一蚕品种个体间的差异进行了研究 .结果发现 :1)在饲料制作上必须进行蒸煮灭菌 ,微波炉加热灭菌与蒸煮的效果相近 ,不经加热杀菌简易化制作的效果都不理想 ;2 )同一蚕品种个体间对人工饲料的摄食性存在差异 .对桑叶粉及蔗糖等摄食促进物质的感受性 ,高食性蚕比低食性蚕敏感 ;而对豆粕粉中摄食阻碍物质的感受性 ,低食性蚕比高食性蚕敏感 .研究认为 ,要提高人工饲料育蚕的群体摄食性和发育整齐度 。

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.

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。

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.

家蚕(Bombyx mori)食性的改变

一、X—幅射诱导食性突变田岛于1952年开始的实验是选用一些生产用品种,在蛹的后期,对雌蛹和雄蛹进行辐射处理,采用的x-射线剂量为6～8KR（千伦琴）。处理蛹化蛾后,使与无处理的配偶蛾进行交配。检验其杂种一代能否喂饲甜菜叶。在三龄蚕中检出变种的机率为11/937,910（0.001％）。但只有一只雄蛾存活,因为甜菜叶带有毒性。这只幸存的雄蛾与同系无处理的雌蛾交配,其F1代显示正常的食性。

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.

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.

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.

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.

家蚕全茧量及重要相关经济性状的多重区间作图分析

利用家蚕品系C100和大造的回交一代BC1群体,用WinQTLCart2.0软件对家蚕全茧量、茧层量、茧层率和蛹体重等4个重要经济性状在分子连锁图的基础上进行了多重区间作图分析。共检测到40个QTL,分布于21个连锁群,其中全茧量、茧层量、茧层率和蛹体重的QTL中,贡献率超过20%的主效QTL数分别为2、2、3、2个,贡献率超过30%的主效QTL有4个,分别是控制全茧量的qCW-19、茧层量的qSW-2、茧层率的qCSR-4和蛹体重的qPW-23。相关显著的经济性状的QTL具有集中分布的特点,并且检测到的QTL均与其单侧标记紧密连锁,利用紧密连锁的分子标记将不同的增效QTL进行聚合,为优质高产家蚕新品种的选育打下基础。

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.

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.